Synergistic antitumour activity of sorafenib in combination with tetrandrine is mediated by reactive oxygen species (ROS)/Akt signaling

Abstract

Background: Sorafenib is a potent inhibitor against Raf kinase and several receptor tyrosine kinases that has been approved for the clinical treatment of advanced renal and liver cancer. Combining sorafenib with other agents has been shown to improve its antitumour efficacy by not only reducing the toxic side effects but also preventing primary and acquired resistance to sorafenib. We have previously observed that tetrandrine exhibits potent antitumour effects in human hepatocellular carcinoma. In this study, we investigated the synergistic antitumour activity of sorafenib in combination with tetrandrine.

Methods: This was a two-part investigation that included the in vitro effects of sorafenib in combination with tetrandrine on cancer cells and the in vivo antitumour efficacy of this drug combination on tumour xenografts in nude mice.

Results: Combined treatment showed a good synergistic antitumour effect yet spared non-tumourigenic cells. The potential molecular mechanism may be mainly that it activated mitochondrial death pathway and induced caspase-dependent apoptosis in the cancer cells. Accumulation of intracellular reactive oxygen species (ROS) and subsequent activation of Akt may also be involved in apoptosis induction.

Conclusion: The antitumour activity of sorafenib plus tetrandrine may be attributed to the induction of the intrinsic apoptosis pathway through ROS/Akt signaling. This finding provides a novel approach that may broaden the clinical application of sorafenib.

Figure 1

The combination of sorafenib and tetrandrine showed synergistic antitumour activity. Data are representative of values from at least three independent experiments. Cell viability was determined by the trypan blue dye-exclusion assay after treatment for 72 h. (A) Treatment of cancer cells with increasing concentrations of sorafenib (0, 2, 4, 6 μℳ) minimally affected cell viability. (B) No cytotoxic effect was observed with tetrandrine treatment (0, 2, 4, 6 μℳ) alone. (C) Sorafenib (4 μℳ) plus tetrandrine (6 μℳ) strongly decreased the viability of human tumour cell lines (**P<0.01). (D) Sorafenib (4 μℳ) plus tetrandrine (6 μℳ) minimally affected the viability of L02 and HBL100 cell lines. (E) Representative dishes from the colony-formation assay. The clonogenic assay was performed as described in Materials and Methods. The results shown here are representative of three independent experiments.

Figure 2

Sorafenib plus tetrandrine induced caspase-dependent apoptosis in cancer cells. All experiments were conducted after treatment with sorafenib (4 μℳ) and tetrandrine (6 μℳ) for 72 h. (A) Sorafenib and tetrandrine combined to induce cell apoptosis. Apoptotic cells were detected by flow cytometry (**P<0.01). (B) Western blot analysis of PARP, caspase-9, pro-caspase-3 and pro-caspase-8 after cells were treated as described above. GAPDH was used as a loading control (**P<0.01). (C) Cell viability was assessed following a 72-h treatment with sorafenib (4 μℳ) and tetrandrine (6 μℳ) with or without a pre-treatment of 50 μℳ z-VAD-fmk (pan-caspase inhibitor) (**P<0.01). (D) Western blot analysis on whole-cell lysates with antibodies against mitochondrial Bcl-2 family members.

Figure 3

Intracellular ROS generation was involved in cellular apoptosis induced by sorafenib plus tetrandrine. (A) Effects of sorafenib plus tetrandrine on intracellular ROS levels after 72 h of treatment. (B) BEL7402 and HCT116 cells were pretreated with 15 mℳ NAC or 10 mℳ Tiron for 1 h and then with the combination of sorafenib and tetrandrine for 72 h. (C) Cell viability was determined in the presence of 15 mℳ NAC after 1 h of treatment (**P<0.01). Values represent mean±s.d. (N=3). (D) Western blot analysis of PARP and caspase-9 levels.

Figure 4

Mitochondrial depolarisation was necessary in combination treatment-induced apoptosis. Cells were treated with sorafenib (4 μℳ) in combination with tetrandrine (6 μℳ) for 72 h. (A) Mitochondrial membrane potential was determined by Rh123 staining and flow cytometry. (B) BEL7402 cells were treated with sorafenib and tetrandrine alone or in combination with 2 μℳ CsA for 72 h, and cell viability was assessed by the trypan blue dye-exclusion assay (**P<0.01). (C) BEL7402 cells were treated in the presence or absence of 2 μℳ CsA for 72 h and then subjected to flow cytometry analysis of cellular apoptosis (**P<0.01). (D) Western blot analysis of cytochrome c release from mitochondria (**P<0.01). (E) Detection of relative ATP level by ATP Assay Kit according to the manufacturer's protocol on a luminometer. The ATP level of untreated cells was set at 100%.

Figure 5

Combination treatment induced cancer cell apoptosis through inhibition of Akt activation. BEL7402 and HCT116 cells were treated with sorafenib (4 μℳ) and tetrandrine (6 μℳ) for 72 h in all the experiments. (A) Protein lysates from cells treated with sorafenib and tetrandrine were subjected to western blot analysis for Akt kinase (**P<0.01). (B) Akt protein level was assessed by western blot after cells were transfected with either the vehicle plasmid (pUSE) or the constitutively active Akt-expressing plasmid (CA-Akt) in the presence or absence of sorafenib (4 μℳ) and tetrandrine (6 μℳ) (**P<0.01). (C) Cell viability was determined after cells were transfected with the vehicle plasmid (pUSE) or the Akt-expressing plasmid (CA-Akt). (D) Western blot analysis of Akt in BEL7402 and HCT116 cells treated with sorafenib plus tetrandrine after a 1-h pretreatment with 15 mℳ NAC. (E) Cells transfected with pUSE or CA-Akt were incubated with sorafenib (4 μℳ) and tetrandrine (6 μℳ) for 72 h and then intracellular ROS was detected by flow cytometry.

Figure 6

Sorafenib and tetrandrine showed synergistic antitumour activity in the in vivo xenograft model. HCT116 cells were inoculated into mice to establish a tumour model as indicated in Materials and Methods. Mice bearing tumours (7 mice per group) were treated with vehicle, sorafenib (25 mg kg⁻¹), tetrandrine (30 mg kg⁻¹) or sorafenib plus tetrandrine every other day for 3 weeks. (A) Mean tumour volumes at given time points. (B) The weights of extracted tumours are presented on a scatter plot; the bars represent the s.d. *P<0.05. (C) Tumour tissue proteins exacted from HCT116 xenografts were subjected to the MDA assay. *P<0.05. (D) Apoptosis analysis of tumour tissues by TUNEL staining.