Sequence‑dependent effect of sorafenib in combination with natural phenolic compounds on hepatic cancer cells and the possible mechanism of action

Abstract

Sorafenib (Nexavar, BAY43‑9006 or Sora) is the first molecular targeted agent that has exhibited significant therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond well to Sora and novel therapeutic strategies to optimize the efficacy of Sora are urgently required. Plant‑based drugs have received increasing attention owing to their excellent chemotherapeutic and chemopreventive activities; they are also well tolerated, non‑toxic, easily available and inexpensive. It is well known that certain biologically active natural products act synergistically with synthetic drugs used in clinical applications. The present study aimed to investigate whether a combination therapy with natural phenolic compounds (NPCs), including curcumin (Cur), quercetin (Que), kaempherol (Kmf) and resveratrol (Rsv), would allow a dose reduction of Sora without concomitant loss of its effectiveness. Furthermore, the possible molecular mechanisms of this synergy were assessed. The hepatic cancer cell lines Hep3b and HepG2 were treated with Sora alone or in combination with NPCs in concomitant, sequential, and inverted sequential regimens. Cell proliferation, cell cycle, apoptosis and expression of proteins associated with the cell cycle and apoptosis were investigated. NPCs markedly potentiated the therapeutic efficacy of Sora in a sequence‑, type‑, NPC dose‑ and cell line‑dependent manner. Concomitant treatment with Sora and Cur [sensitization ratio (SR)=28], Kmf (SR=18) or Que (SR=8) was associated with the highest SRs in Hep3b cells. Rsv markedly potentiated the effect of Sora (SR=17) on Hep3b cells when administered in a reverse sequential manner. By contrast, Rsv and Que did not improve the efficacy of Sora against HepG2 cells, while concomitant treatment with Cur (SR=10) or Kmf (SR=4.01) potentiated the cytotoxicity of Sora. Concomitant treatment with Sora and Cur or Kmf caused S‑phase and G2/M phase arrest of liver cancer cells and markedly induced apoptosis compared with mono‑treatment with Sora, Cur or Kmf. Concomitant treatment with Sora and Cur reduced the protein levels of cyclins A, B2 and D1, phosphorylated retinoblastoma and B‑cell lymphoma (Bcl) extra‑large protein. By contrast, Sora and Cur co‑treatment increased the protein levels of Bcl‑2‑associated X protein, cleaved caspase‑3 and cleaved caspase‑9 in a dose‑dependent manner. In conclusion, concomitant treatment with Sora and Cur or Kmf appears to be a potent and promising therapeutic approach that may control hepatic cancer by triggering cell cycle arrest and apoptosis. Additional studies are required to examine the potential of combined treatment with Sora and NPCs in human hepatic cancer and other solid tumor types in vivo.

Figure 1

Dose-dependent anti-proliferative effect of (a) Cur, (b) Kmf, (c) Que and (d) Rsv (20-160 μM) for 72 h on (A) CRL1554 normal human fibroblast, and (B) Hep3b and (C) HepG2 hepatic cancer cell lines. Cell growth was monitored using an MTT assay. Cur, curcumin; Kmf, kaempferol; Que, quercetin; Rsv, resveratrol.

Figure 2

Sequence-dependent anti-proliferative effect of combined treatment with Sora and Cur on (A) Hep3b and (B) HepG2 hepatic cancer cell lines. The cells were treated with a combination of Sora (5 μM) and Cur (60 or 120 μM) in (a) a sequential, (b) inverted sequential and (c) concomitant manner. Cell growth was determined by an MTT assay. Sora, sorafenib; Cur, curcumin; UT, untreated, ^*P<0.05 (Sora + 60 μM Cur) vs. Sora, ^**P<0.05 (Sora + 120 μM Cur) vs. Sora, ^***P<0.05 Sora vs. (Sora + 60 μM Cur or 120 μM Cur).

Figure 3

Sequence-dependent anti-proliferative effect of combined treatment with Sora and Kmf on (A) Hep3b and (B) HepG2 hepatic cancer cell lines. The cells were treated with a combination of Sora (5 μM) and Kmf (60 and 120 μM) in (a) a sequential, (b) inverted sequential and (c) concomitant manner. Cell growth was determined by an MTT assay. Kmf, kaempferol; Sora, sorafenib; UT, untreated, ^**P<0.05 (Sora + 120 μM Kmf) vs. Sora, ^***P<0.05 Sora vs. (Sora + 60 μM Kmf).

Figure 4

Sequence-dependent anti-proliferative effect of combined treatment Sora and Que on (A) Hep3b and (B) HepG2 hepatic cancer cell lines. The cells were treated with a combination of Sora (5 μM) and Que (60 and 120 μM) in (a) a sequential, (b) inverted sequential and (c) concomitant manner. Cell growth was determined by an MTT assay. Que, quercetin; Sora, sorafenib; UT, untreated, ^**P<0.05 (Sora + 120 μM Que) vs. Sora, ^***P<0.05 Sora vs. (Sora + 60 μM Que).

Figure 5

Sequence-dependent anti-proliferative effect of combined treatment with Sora and Rsv on (A) Hep3b and (B) HepG2 hepatic cancer cell lines. The cells were treated with a combination of Sora (5 μM) and Rsv (40 or 80 μM) in (a) a sequential, (b) inverted sequential and (c) concomitant manner. Cell growth was monitored by MTT assay. Sora, sorafenib; UT, untreated; Rsv, resveratrol, ^*P<0.05 (Sora + 40 μM Rsv) vs. Sora, ^**P<0.05 (Sora + 80 μM Rsv) vs. Sora, ^***P<0.05 Sora vs. (Sora + 40 μM Rsv or 80 μM Rsv).

Figure 6

Analysis of cell cycle perturbation in human cancer cell lines treated with Sora, Cur and their simultaneous combination. (A) Hep3b and (B) HepG2 hepatic cancer cell lines were either left untreated or treated with Sora (5 μM), Cur (200 μM), Cur (400 μM), or simultaneous combinations of Sora and Cur (5 μM + 200 μM) or (5 μM + 400 μM) for 72 h. At least three samples were analyzed and 20,000 events were scored for each sample. The vertical axis represents the relative number of events and the horizontal axis represents the fluorescence intensity. Sora, sorafenib; Cur, curcumin; UT, untreated. The black and white curves are for control and experimental groups, respectively.

Figure 7

Analysis of cell cycle perturbation in human cancer cell lines treated with Sora, Kmf and their simultaneous combination. (A) Hep3b and (B) HepG2 hepatic cancer cell lines were either left untreated or treated with Sora (5 μM), Kmf (200 μM), Kmf (400 μM), or simultaneous combinations of Sora and Kmf (5 μM + 200 μM) or (5 μM + 400 μM) for 72 h. At least three samples were analyzed and 20,000 events were scored for each sample. The vertical axis represents the relative number of events and the horizontal axis represents the fluorescence intensity. The black and white curves are for control and experimental groups, respectively. Sora, sorafenib; Kmf, kaempferol; UT, untreated.

Figure 8

Analysis of DNA fragmentation in human hepatic cancer cell lines treated with Sora, Cur, Kmf and their simultaneous combinations. (A and B) Hep3b and HepG2 hepatic cancer cell lines treated with Sora (5 μM), Cur (200 or 400 μM), or their simultaneous combinations or (C and D) Sora (5 μM), Kmf (200 or 400 μM) and their simultaneous combinations for 48 h. DNA fragments were extracted and analyzed on 1% agarose. M, 1,000 bp DNA marker. Sora, sorafenib; Kmf, kaempferol; UT, untreated; Cur, curcumin.

Figure 9

Flow cytometric analysis of apoptosis of human hepatic cancer cell lines treated with Sora, Cur and their simultaneous combinations. (A) Hep3b and (B) HepG2 hepatic cancer cell lines were (a) left untreated or treated with (b) Sora (5 μM), (c) Cur (200 μM), (d) Cur (400 μM), or (e and f) simultaneous combinations of Sora and Cur (5 μM + 200 μM, respectively) or (5 μM + 400 μM, respectively) for 48 h. The cells were analyzed by flow cytometry after processing and staining with Annexin V-FITC/PI. B1, percentage of necrotic cells; B2, percentage of late apoptotic cells; B3, percentage of viable cells; and B4, percentage of early apoptotic cells. Sora, sorafenib; Cur, curcumin; UT, untreated; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 10

Flow cytometric analysis of apoptosis in human hepatic cancer cell lines treated with Sora, Kmf and their simultaneous combinations. (A) Hep3b and (B) HepG2 hepatic cancer cell lines were (a) left untreated or treated with (b) Sora (5 μM), (c) Kmf (200 μM), (d) Kmf (400 μM), or (e and f) simultaneous combinations of Sora and Kmf (5 μM + 200 μM, respectively) or (5 μM + 400 μM, respectively) for 48 h. The cells were analyzed by flow cytometry after processing and staining with Annexin V-FITC/PI. B1, percentage of necrotic cells; B2, percentage of late apoptotic cells; B3, percentage of viable cells; and B4, percentage of early apoptotic cells. Sora, sorafenib; Kmf, kaempferol; UT, untreated; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 11

Flow cytometric analysis of MMP in human hepatic cancer cell lines treated with Sora, Cur and their simultaneous combinations. (A) Hep3b and (B) HepG2 hepatic cancer cell lines were (a) left untreated or treated with (b) Sora (5 μM), (c) Cur (200 μM), (d) Cur (400 μM), or (e and f) simultaneous combinations of Sora and Cur (5 μM + 200 μM, respectively) or (5 μM + 400 μM, respectively) for 48 h. Alterations in MMP were monitored by staining with the MitoNIR dye and flow cytometric analysis with excitation/emission wavelengths of 635 and 660 nm, respectively. The red and white curves are the control and experimental groups, respectively. MMP, mitochondrial membrane potential; Sora, sorafenib; Cur, curcumin.

Figure 12

Flow cytometric analysis of MMP in human hepatic cancer cell lines treated with Sora, Kmf and their simultaneous combinations. (A) Hep3b and (B) HepG2 hepatic cancer cell lines were (a) left untreated or treated with (b) Sora (5 μM), (c) Kmf (200 μM), (d) Kmf (400 μM), or (e and f) simultaneous combinations of Sora and Kmf (5 μM + 200 μM, respectively) or (5 μM + 400 μM, respectively) for 48 h. Alterations in MMP were monitored by staining with the MitoNIR dye and flow cytometric analysis with excitation/emission wavelengths of 635 and 660 nm, respectively. Alterations in MMP were monitored by staining with the MitoNIR dye and flow cytometric analysis with excitation/emission wavelengths of 635 and 660 nm, respectively. The red and white curves are the control and experimental groups, respectively. MMP, mitochondrial membrane potential; Sora, sorafenib; Kmf, kaempferol.

Figure 13

Western blot analysis of the levels of cell cycle and apoptosis-associated proteins in the extract of human hepatic cancer cell lines Hep3b and HepG2 treated with Sora, Cur, and their combinations. The cells were treated with Sora (5 μM), Cur (200 μM), Cur (400 μM) or a simultaneous combination of Sora and Cur (5 μM + 200 or 400 μM) for 48 h. The levels of proteins associated with the control of cell cycle and apoptosis were analyzed by western blot analysis. Signal intensities of the respective bands were densitometrically quantified. (A) Cell cycle-associated proteins and (B) apoptosis-associated proteins. Sora, sorafenib; Cur, curcumin; UT, untreated; pRb, retinoblastoma protein; Bcl-xL, B-cell lymphoma extra-large protein; Bax, B-cell lymphoma 2-associated X protein.