Due to unsatisfactory outcomes of available chemotherapies, new therapeutic strategies for HCC are urgently needed. More effective treatments may involve combination of agents with synergistic activity against HCC. Rapamycin, a mTOR inhibitor, has been used in treating various solid tumors. Despite its outstanding preclinical antitumor activity, rapamycin has been shown to increase Akt phosphorylation, which may counteract its anticancer efficacy [14
] And could be a reason why mTOR inhibitors only exhibit modest antitumor activity in patients. In this study, we hypothesized that the combined use of an agent which down-regulates Akt survival pathway would potentiate the antitumor effects of rapamycin in HCC cells. Focus on the activation of Akt caused by mTOR inhibition, there are some novel therapeutic approaches [32
]. In our study, we chose the combination of rapamycin with bortezomib for a couple of reasons. Firstly, rapamycin and bortezomib have shown single-agent activity in preclinical studies in HCC. Secondly, PI3K/Akt/mTOR pathway is critical for cell survival and resistance to apoptosis and both agents interact at the PI3K/Akt/mTOR pathway [10
], which is known to be activated in HCC. Our results in this study have demonstrated that suppression of mTOR signaling by rapamycin in HCCLM3 cells was associated with up-regulation of Akt phosphorylation, which was abrogated by bortezomib in a dose-dependent manner. These in vitro
results were also confirmed in vivo
, as bortezomib significantly suppressed rapamycin-induced activation of Akt, which in turn enhanced the rapamycin-induced inhibition of tumor growth and pulmonary pulmonary metastasis in orthotopic HCC mice. These data suggested that it is rational to combine rapamycin with bortezomib for targeted HCC therapy.
Although rapamycin has been previously reported to induce cell apoptosis [35
], rapamycin treatment alone did not induce apoptosis of HCCLM3 and SMMC7721 cells in this study. However, we demonstrated that the combination of rapamycin and bortezomib produced greater apoptotic activity in HCCLM3 and SMMC7721 cells than either agent alone. This synergistic apoptotic effect was at least partially due to the suppression of rapamycin-induced Akt activation by bortezomib, which may overcome rapamycin resistance. In addition, our data demonstrated that the combination of rapamycin and bortezomib significantly enhanced the expression of p53 mRNA, which may lead to an irreversible apoptotic commitment. The activation of p53 may also contribute to apoptosis by inhibiting Akt phosphorylation [36
]. Once activated, p53 induces up-regulation of p21 and Bax, which mediate different aspects of p53 function on proliferation arrest and apoptosis [38
]. Our data suggested that the pro-apoptotic effect of rapamycin plus bortezomib involves elevated transcriptional activity of p53. P53 inhibitor PFT-α can attenuate the pro-apoptotic effect of the combined treatment. Based on our results, we propose that the combined treatment induced apoptosis in HCCLM3 cells is mainly, if not completely, dependent on p53 activity.
The expression of p27 mRNA in HCCLM3 cells was also significantly up-regulated by rapamycin or the combination of rapamycin and bortezomib. Moss et al.
reported that rapamycin increased p27 levels, which inhibited migration of human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAEC). Silencing of p27 with small interfering RNA blocked the effects of rapamycin on migration and tube formation [40
]. Although the link between mTOR inhibition and cell migration need further investigation, the increased level of p27 induced by rapamycin or the combination treatment may be a potential molecular mechanism involved in the regulation of HCCLM3 cell migration.
Furthermore, we examined the effects of rapamycin and/or bortezomib in orthotopic tumor model of HCC. Rapamycin alone significantly inhibited HCCLM3-R tumor growth. Bortezomib produced less growth inhibition in HCCLM3-R tumors as compared to rapamycin, even though bortezomib induced higher level of apoptosis in HCCLM3 cells in vitro
. The combination of rapamycin and bortezomib generated greater tumor growth inhibition than either agent alone, with the rate of tumor growth inhibition index as high as 72.4%. Moreover, only 16.6% of mice (1/6) developed pulmonary metastasis in the combination treatment group. Our data demonstrated that bortezomib down-regulated the rapamycin-induced activation of Akt in tumor tissues, which may be one of mechanisms of the novel synergistic antitumor effects. The average proliferation index was dramatically decreased from 62.8
12.7% in rapamycin treatment group to 29.8
6.5% in the group that received both rapamycin and bortezomib. Rapamycin has showed antiangiogenic activities in vivo
not only by decreasing the production of VEGF but also by inhibiting the response of vascular endothelial cells on stimulation by VEGF via
inhibiting of mTOR [41
]. One mechanism of bortezomib-induced tumor growth inhibition identified in previous studies is also angiogenesis inhibition [42
]. In our study, rapamycin, but not bortezomib, significantly reduced microvessel density in orthotopic HCC tumors. The combination of rapamycin and bortezomib amplified the rapamycin-induced inhibition of tumor angiogenesis from 33.6% to 53.0%.The mechanism of synergistic inhibition of angiogenesis by these agents is still unclear and needs further investigation.
In this study, we demonstrated the synergistic effects of rapamycin and bortezomib in inhibiting cell growth, inducing cell apoptosis, and suppressing cell migration and invasion in vitro. Rapamycin alone or in combination with bortezomib, strongly inhibited HCC growth and pulmonary metastasis in vivo. Bortezomib overcame rapamycin resistance in HCC cells partly through inhibition of the PI3K/Akt pathway activated by rapamycin. In summary, this study provides preclinical evidence that the combination of rapamycin and bortezomib could be a novel and promising therapeutic approach to the treatment of HCC, which warrants further investigation in a clinical setting.