3.1. Brusatol synergistically enhanced growth inhibition caused by lapatinib in SK-BR-3, SK-OV-3 and AU565 cells
In the previous study, we found that brusatol exerted anti-tumor effects against HER2-overexpressed cancer cells by repressing HER2-AKT/ERK1/2 signaling pathway . Based on these results, we hypothesize that addition of brusatol to lapatinib may result in a therapeutic benefit in treating HER2-positive cancers. Results revealed that lapatinib in combination with brusatol exhibited an significantly enhanced inhibitory activity than that of either agent alone in all three HER2-positive cancer cells including SK-BR-3, SK-OV-3 and AU565 cancer cells (Fig 1A). Moreover, the superior effects of lapatinib plus brusatol was synergistic on SK-BR-3, SK-OV-3 and AU-565 cells by utilizing the method of Chou and Talalay to establish drug C.I. values (Fig 1B).
3.2. Brusatol sensitizes HER2-positive cells to lapatinib by inducing the up-regulation of the ROS level in SK-BR-3 and SK-OV-3 cancer cells
Recent studies illustrated that brusatol might provide potential clinical benefit especially when combined with anti-tumor drugs that stimulate ROS production. [17, 20]. Therefore, we also examined the effects of combinatorial treatment on ROS accumulation utilizing FACS assay. Results revealed that co-treatment of lapatinib with brusatol resulted in a significantly greater increase on ROS production than brusatol or lapatinib treatment alone in both SK-OV-3 and SK-BR-3 cancer cells (Fig 2). To further provide additional evidence for these results, N-acetyl-L-cysteine (NAC), a type of anti-oxidant agent that can effectively reduce the ROS level was added to SK-OV-3 and SK-BR-3 cells treated with a combination of lapatinib and brusatol. As expected, the co-administration of the anti-oxidant NAC antagonized the elevation in ROS production from both SK-OV-3 and SK-BR-3 cell lines treated with brusatol plus lapatinib (Fig 2). To conclude, brusatol plus lapatinib was more effective in inducing ROS production than either agent alone, which may explain the superior anti-tumor effects of the combinatorial treatment.
3.3. Brusatol in combination with lapatinib potently potentiated apoptosis in SK-BR-3 and SK-OV-3 cells
As we know, Nrf2 inhibition by Nrf2-targeted agents renders cancer cells susceptible to apoptosis [19, 21]. Thus, we evaluated the apoptotic percentage in SK-BR-3 and SK-OV-3 cells when were exposed to brusatol plus lapatinib. Compared to brusatol or lapatinib alone, a combination of brusatol with lapatinib significantly potentiated apoptosis in SK-BR-3 cells (Fig 3A and B). Notably, as shown in Figure 3C and D, combinatorial therapy has not exhibited the superiority in SK-OV-3 cells compared to brusatol group, which suggested that apoptosis activation may be not the main mechanism explaining the superior growth-inhibitory effects in combinatorial group.
3.4. Lapatinib plus brusatol exhibited a significant effect on regressing Nrf2/HO-1 anti-oxidant and EGFR/HER2-AKT/ERK1/2 signaling pathways in SK-BR-3 and SK-OV-3 cancer cells
To further explore the mechanism involved in synergistic anti-tumor effect, we examined the core protein level involved in Nrf2/HO-1 signaling and EGFR/HER2-AKT/ERK1/2 signaling pathway in both SK-BR-3 and SK-OV-3 cancer cells. Western blot analysis showed that brusatol in combination with lapatinib was more effective in inhibiting phosphorylation of EGFR and HER2 than either single-drug treatment. Moreover, weaker phosphorylation level of AKT and MAPK was observed in the two cell lines treated with contaminant treatment, respectively, compared with single agent treatments (Fig 4). Notably, nearly complete AKT deactivation was observed in the condition of contaminant treatment compared with single-agent treatments, while ERK1/2 activation was only partly inhibited (Fig 4). It suggested that the combinatorial treatment may mainly preclude HER2/EGFR-AKT signaling activation, thereby resulted in cell growth repression.
3.5. Combination therapy of lapatinib and brusatol is superior to single-agent treatment in SK-OV-3 xenografts
To further evaluate the therapeutic effects in vivo, brusatol plus lapatinib was injected in nude mice bearing established SK-OV-3 xenograft tumors. Nude mice bearing SK-OV-3 xenografts were randomized and treated either with lapatinib (100 mg/kg), brusatol (2 mg/kg), or both. Results showing that contaminant treatment of brusatol and lapatinib resulted in a significant benefit over either brusatol or lapatinib alone in SK-OV-3 xenografts (Fig 5A). Meanwhile, we found that the mice in combinatorial group did not significantly lose body weight and behaved normally in the treatment period (Fig 5B). As is shown in Figure 5C and 5D, the treatment with lapatinib and brusatol combination resulted in a 50 % reduction in tumor weight in comparison with control. Besides these, hematoxylin and eosin (H&E) staining also showed that no marked liver toxicity was observed in SK-OV-3 tumor-bearing mice upon treatment with lapatinib plus brusatol (Fig S1). Overall, these results above revealed that the combinatorial therapy shows stronger inhibitory effects and appear to be well tolerated in HER2-positive tumor xenografts.