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The synergistic in vitro and in vivo antitumor effect of combination therapy with salinomycin and 5-fluorouracil against hepatocellular carcinoma.

Wang F, Dai W, Wang Y, Shen M, Chen K, Cheng P, Zhang Y, Wang C, Li J, Zheng Y, Lu J, Yang J, Zhu R, Zhang H, Zhou Y, Xu L, Guo C - PLoS ONE (2014)

Bottom Line: The combination of Sal and 5-FU resulted in a synergistic antitumor effect against liver tumors both in vitro and in vivo.Sal reversed the 5-FU-induced increase in CD133(+) EPCAM(+) cells, epithelial-mesenchymal transition and activation of the Wnt/β-catenin signaling pathway.The combination of Sal and 5-FU may provide us with a new approach to reverse drug resistant for the treatment of patients with HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Shanghai Tenth People's Hospital, Tongji University of Medicine, Shanghai, PR China.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the few cancers in which a continuous increase in incidence has been observed over several years. Drug resistance is a major problem in the treatment of HCC. In the present study, we used salinomycin (Sal) and 5-fluorouracil (5-FU) combination therapy on HCC cell lines Huh7, LM3 and SMMC-7721 and nude mice subcutaneously tumor model to study whether Sal could increase the sensitivity of hepatoma cells to the traditional chemotherapeutic agent such as 5-FU. The combination of Sal and 5-FU resulted in a synergistic antitumor effect against liver tumors both in vitro and in vivo. Sal reversed the 5-FU-induced increase in CD133(+) EPCAM(+) cells, epithelial-mesenchymal transition and activation of the Wnt/β-catenin signaling pathway. The combination of Sal and 5-FU may provide us with a new approach to reverse drug resistant for the treatment of patients with HCC.

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Combination treatment with 5-FU and Sal.(A–D) Illustrative Fa-CI and Fa-DRI plots for the combination of 5-FU and Sal using different fixed drug ratios. (A) CI values were calculated from each Fa for HCC cell lines Huh7, LM3, and SMMC-7721. Average synergism (CI<1) at Fa>0.5 for all three HCC lines. (B) DRI values were calculated from each Fa for HCC cell lines Huh7, LM3, and SMMC-7721. The 5-FU and Sal chemotherapeutic doses may be significantly reduced (DRI>1) for combinations that are synergistic at Fa>0.5 for all three HCC lines. (C) Isobologram analysis at IC50, IC60 and IC70 for the combinations of HCC cell lines Huh7, LM3, and SMMC-7721. The results indicats synergy, additivity or antagonism when the points are located below, on or above the line, respectively. We can see that (C5-FU, CSal) is located below the line (synergy) at IC 60, IC 70 for HCC cell lines Huh7, LM3 and SMMC-7721. (D) The combination effect of Sal and 5-FU on apoptosis effects were evaluated by flow cytometric analysis. The results showed that combination therapy increased apoptosis of HCC cell lines Huh7, LM3 and SMMC-7721 significantly. (E–R) Combination treatments in the in vivo models (E) Subcutaneous tumor volume following combination therapy was reduced compared to that of the other three groups (two representative mice in each group). (F) HE staining showed the area of apoptosis and necrosis induced by drugs in tumor tissue of treatment group. (G) The tumor growth curve showed that tumor growth rate following combination therapy was slower than that of the other three groups. (H) The relative tumor proliferation rate, VTreatment/VControl, showed that proliferation rate of the combination therapy group was slower than that of the other three groups. (*p<0.05)s. (I) In the combination therapy group, tumor blocks weighed lighter than those of the other three groups (*p<0.05). (R) The tumor growth inhibition rate indicated that the combination therapy significantly inhibited tumor growth than the other three groups (*p<0.05).
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pone-0097414-g002: Combination treatment with 5-FU and Sal.(A–D) Illustrative Fa-CI and Fa-DRI plots for the combination of 5-FU and Sal using different fixed drug ratios. (A) CI values were calculated from each Fa for HCC cell lines Huh7, LM3, and SMMC-7721. Average synergism (CI<1) at Fa>0.5 for all three HCC lines. (B) DRI values were calculated from each Fa for HCC cell lines Huh7, LM3, and SMMC-7721. The 5-FU and Sal chemotherapeutic doses may be significantly reduced (DRI>1) for combinations that are synergistic at Fa>0.5 for all three HCC lines. (C) Isobologram analysis at IC50, IC60 and IC70 for the combinations of HCC cell lines Huh7, LM3, and SMMC-7721. The results indicats synergy, additivity or antagonism when the points are located below, on or above the line, respectively. We can see that (C5-FU, CSal) is located below the line (synergy) at IC 60, IC 70 for HCC cell lines Huh7, LM3 and SMMC-7721. (D) The combination effect of Sal and 5-FU on apoptosis effects were evaluated by flow cytometric analysis. The results showed that combination therapy increased apoptosis of HCC cell lines Huh7, LM3 and SMMC-7721 significantly. (E–R) Combination treatments in the in vivo models (E) Subcutaneous tumor volume following combination therapy was reduced compared to that of the other three groups (two representative mice in each group). (F) HE staining showed the area of apoptosis and necrosis induced by drugs in tumor tissue of treatment group. (G) The tumor growth curve showed that tumor growth rate following combination therapy was slower than that of the other three groups. (H) The relative tumor proliferation rate, VTreatment/VControl, showed that proliferation rate of the combination therapy group was slower than that of the other three groups. (*p<0.05)s. (I) In the combination therapy group, tumor blocks weighed lighter than those of the other three groups (*p<0.05). (R) The tumor growth inhibition rate indicated that the combination therapy significantly inhibited tumor growth than the other three groups (*p<0.05).

Mentions: The effect of Sal combined with 5-FU on cell viability was investigated using the MTT assay. For these studies, HCC cell lines Huh7, LM3 and SMMC-7721 were treated with 5-FU (0, 2, 4, 8, and 16 ug/ml), Sal (0, 2, 4, 8, and 16 µM), or Sal plus 5-FU for 48 h. Viable cells were evaluated using the MTT assay. Treatment of HCC cells with 5-FU plus Sal for 48 h resulted in a decrease in cell viability which was greater than either 5-FU or Sal alone (Table 2). Fraction affected (Fa) values (indicating the fraction of cells inhibited after drug exposure) were obtained after exposure of the cells to a series of drug concentrations. To indicate the effects at different Fa values, the CI (combination index) and DRI (dose reduction index) values were calculated for each Fa. Fig. 2A shows the Fa-CI plots illustrating the effects of Sal and 5-FU at different fixed drug ratios, and demonstrates synergism (CI<1) at Fa>0.5 for HCC cell lines Huh7, LM3 and SMMC-7721. As expected, synergism corresponding to CI<1 always yielded a favorable DRI (>1) for both drugs. The Fa-DRI plots are shown in Fig. 2B, and indicate that chemotherapeutic doses of 5-FU may be significantly reduced for combinations with Sal that are synergistic at Fa>0.5. Classical isobolograms were shown in Fig. 2C, we can see that (C5-FU, CSal) is located below the line (synergy) at IC60, IC70 for HCC cell lines Huh7, LM3 and SMMC-7721. At last the combination effect of Sal and 5-FU on apoptosis effects were evaluated by flow cytometric analysis. The results (Fig. 2D) showed that combination therapy increased apoptosis of HCC cell lines Huh7, LM3 and SMMC-7721 significantly.


The synergistic in vitro and in vivo antitumor effect of combination therapy with salinomycin and 5-fluorouracil against hepatocellular carcinoma.

Wang F, Dai W, Wang Y, Shen M, Chen K, Cheng P, Zhang Y, Wang C, Li J, Zheng Y, Lu J, Yang J, Zhu R, Zhang H, Zhou Y, Xu L, Guo C - PLoS ONE (2014)

Combination treatment with 5-FU and Sal.(A–D) Illustrative Fa-CI and Fa-DRI plots for the combination of 5-FU and Sal using different fixed drug ratios. (A) CI values were calculated from each Fa for HCC cell lines Huh7, LM3, and SMMC-7721. Average synergism (CI<1) at Fa>0.5 for all three HCC lines. (B) DRI values were calculated from each Fa for HCC cell lines Huh7, LM3, and SMMC-7721. The 5-FU and Sal chemotherapeutic doses may be significantly reduced (DRI>1) for combinations that are synergistic at Fa>0.5 for all three HCC lines. (C) Isobologram analysis at IC50, IC60 and IC70 for the combinations of HCC cell lines Huh7, LM3, and SMMC-7721. The results indicats synergy, additivity or antagonism when the points are located below, on or above the line, respectively. We can see that (C5-FU, CSal) is located below the line (synergy) at IC 60, IC 70 for HCC cell lines Huh7, LM3 and SMMC-7721. (D) The combination effect of Sal and 5-FU on apoptosis effects were evaluated by flow cytometric analysis. The results showed that combination therapy increased apoptosis of HCC cell lines Huh7, LM3 and SMMC-7721 significantly. (E–R) Combination treatments in the in vivo models (E) Subcutaneous tumor volume following combination therapy was reduced compared to that of the other three groups (two representative mice in each group). (F) HE staining showed the area of apoptosis and necrosis induced by drugs in tumor tissue of treatment group. (G) The tumor growth curve showed that tumor growth rate following combination therapy was slower than that of the other three groups. (H) The relative tumor proliferation rate, VTreatment/VControl, showed that proliferation rate of the combination therapy group was slower than that of the other three groups. (*p<0.05)s. (I) In the combination therapy group, tumor blocks weighed lighter than those of the other three groups (*p<0.05). (R) The tumor growth inhibition rate indicated that the combination therapy significantly inhibited tumor growth than the other three groups (*p<0.05).
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Related In: Results  -  Collection

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pone-0097414-g002: Combination treatment with 5-FU and Sal.(A–D) Illustrative Fa-CI and Fa-DRI plots for the combination of 5-FU and Sal using different fixed drug ratios. (A) CI values were calculated from each Fa for HCC cell lines Huh7, LM3, and SMMC-7721. Average synergism (CI<1) at Fa>0.5 for all three HCC lines. (B) DRI values were calculated from each Fa for HCC cell lines Huh7, LM3, and SMMC-7721. The 5-FU and Sal chemotherapeutic doses may be significantly reduced (DRI>1) for combinations that are synergistic at Fa>0.5 for all three HCC lines. (C) Isobologram analysis at IC50, IC60 and IC70 for the combinations of HCC cell lines Huh7, LM3, and SMMC-7721. The results indicats synergy, additivity or antagonism when the points are located below, on or above the line, respectively. We can see that (C5-FU, CSal) is located below the line (synergy) at IC 60, IC 70 for HCC cell lines Huh7, LM3 and SMMC-7721. (D) The combination effect of Sal and 5-FU on apoptosis effects were evaluated by flow cytometric analysis. The results showed that combination therapy increased apoptosis of HCC cell lines Huh7, LM3 and SMMC-7721 significantly. (E–R) Combination treatments in the in vivo models (E) Subcutaneous tumor volume following combination therapy was reduced compared to that of the other three groups (two representative mice in each group). (F) HE staining showed the area of apoptosis and necrosis induced by drugs in tumor tissue of treatment group. (G) The tumor growth curve showed that tumor growth rate following combination therapy was slower than that of the other three groups. (H) The relative tumor proliferation rate, VTreatment/VControl, showed that proliferation rate of the combination therapy group was slower than that of the other three groups. (*p<0.05)s. (I) In the combination therapy group, tumor blocks weighed lighter than those of the other three groups (*p<0.05). (R) The tumor growth inhibition rate indicated that the combination therapy significantly inhibited tumor growth than the other three groups (*p<0.05).
Mentions: The effect of Sal combined with 5-FU on cell viability was investigated using the MTT assay. For these studies, HCC cell lines Huh7, LM3 and SMMC-7721 were treated with 5-FU (0, 2, 4, 8, and 16 ug/ml), Sal (0, 2, 4, 8, and 16 µM), or Sal plus 5-FU for 48 h. Viable cells were evaluated using the MTT assay. Treatment of HCC cells with 5-FU plus Sal for 48 h resulted in a decrease in cell viability which was greater than either 5-FU or Sal alone (Table 2). Fraction affected (Fa) values (indicating the fraction of cells inhibited after drug exposure) were obtained after exposure of the cells to a series of drug concentrations. To indicate the effects at different Fa values, the CI (combination index) and DRI (dose reduction index) values were calculated for each Fa. Fig. 2A shows the Fa-CI plots illustrating the effects of Sal and 5-FU at different fixed drug ratios, and demonstrates synergism (CI<1) at Fa>0.5 for HCC cell lines Huh7, LM3 and SMMC-7721. As expected, synergism corresponding to CI<1 always yielded a favorable DRI (>1) for both drugs. The Fa-DRI plots are shown in Fig. 2B, and indicate that chemotherapeutic doses of 5-FU may be significantly reduced for combinations with Sal that are synergistic at Fa>0.5. Classical isobolograms were shown in Fig. 2C, we can see that (C5-FU, CSal) is located below the line (synergy) at IC60, IC70 for HCC cell lines Huh7, LM3 and SMMC-7721. At last the combination effect of Sal and 5-FU on apoptosis effects were evaluated by flow cytometric analysis. The results (Fig. 2D) showed that combination therapy increased apoptosis of HCC cell lines Huh7, LM3 and SMMC-7721 significantly.

Bottom Line: The combination of Sal and 5-FU resulted in a synergistic antitumor effect against liver tumors both in vitro and in vivo.Sal reversed the 5-FU-induced increase in CD133(+) EPCAM(+) cells, epithelial-mesenchymal transition and activation of the Wnt/β-catenin signaling pathway.The combination of Sal and 5-FU may provide us with a new approach to reverse drug resistant for the treatment of patients with HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Shanghai Tenth People's Hospital, Tongji University of Medicine, Shanghai, PR China.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the few cancers in which a continuous increase in incidence has been observed over several years. Drug resistance is a major problem in the treatment of HCC. In the present study, we used salinomycin (Sal) and 5-fluorouracil (5-FU) combination therapy on HCC cell lines Huh7, LM3 and SMMC-7721 and nude mice subcutaneously tumor model to study whether Sal could increase the sensitivity of hepatoma cells to the traditional chemotherapeutic agent such as 5-FU. The combination of Sal and 5-FU resulted in a synergistic antitumor effect against liver tumors both in vitro and in vivo. Sal reversed the 5-FU-induced increase in CD133(+) EPCAM(+) cells, epithelial-mesenchymal transition and activation of the Wnt/β-catenin signaling pathway. The combination of Sal and 5-FU may provide us with a new approach to reverse drug resistant for the treatment of patients with HCC.

Show MeSH
Related in: MedlinePlus