Experimental Study On The Mechanism And Intervention Of Sorafenib In Inhibiting The Metastasis Of Natural Killer Cells (NK)

Hepatocellular carcinoma (HCC) is the fifth-most prevalent malignant tumor worldwide. Although surgical resection and liver transplantation are the main modalities of curative treatment for HCC, most patients present late stages of the disease, when curative treatment is not feasible and outcomes are likely to be poor. Although nonsurgical treatments for HCC are available, such as radiofrequency ablation and transcatheter arterial chemoembolisation, the overall survival rate is not satisfactory. In recent years, molecular targeted therapy have demonstrated a survival benefit. Sorafenib was the first molecularly targeted agent approved for treating advanced HCC. Although the SHARP and ORIENTAL trials demonstrated sorafenib’s survival benefit, its efficacy is only moderate, because the survival benefit is less than three months.The present study was undertaken to determine whether the growth and metastasis of HCC were influenced in mice receiving sorafenib prior to implantation with tumors and demonstrated that the off-target effects of sorafenib on host immunity. Furthermore, we investigated the underlying mechanism and explored clinical available approach to improve the effect of sorafenib in HCC.1. Sorafenib pretreatment promoted metastatic potential of HCC in nude mice and immunocompetent miceFirstly, pretreatment with vehicle or sustained sorafenib was initiated2weeks and was stopped24hours before the implantation of different tumors in T cell deficient and NK cells intact nude mice. Next, we examined the potential of growth and metastases of tumor cells and survial in sorafenib-pretreated nude mice. We found sorafenib pretreatment (60mg·kg-1day-1,2weeks) significantly promoted tumor growth, compared to the vehicle-pretreated nude mice in LM3subcutaneous tumor model. In tail vein injected HepG2-GFP tumor cells models, more lung metastatic foci were found in the sorafenib-pretreated nude mice, and survival time was significantly shorter in sorafenib-pretreated nude mice, compared with vehicle-pretreated nude mice. The number of lung metastasis was significantly increased by sorafenib pretreatment in the LM3-RFP orthotopic model, although the difference in tumor volume was not statistically significant.To verify the above findings in immunocompetent mice, we established experimental lung metastasis in C57BL/6mice with mouse HCC cell line Hepal-6-GFP. Again, enhanced lung metastasis was observed in sorafenib-pretreated (60mg-kg--1day-1) mice but not in vehicle-pretreated C57BL/6mice.2. Suppression of natural killer cells by sorafenib contributes to prometastatic effects in hepatocellular carcinomaAfter treating the mice for2weeks with2different doses of sorafenib, we examined the number of T cells and NK cells in the spleen and peripheral blood in orthotopic Hepal-6tumor-bearing C57BL/6mice and tumor-free C57BL/6mice. Sorafenib decreased the number of NK cells in a dose-dependent manner in both tumor-bearing and tumor-free mice. However, the changes of CD4+T cells and CD8+T cells in tumor-bearing mice were not significant. The expression of activated mark CD69in NK cells was downregulated by sorafenib (30mg·kg-1day-1and60mg·kg-1day-1,2weeks) in tumor-bearing C57BL/6mice but not in tumor-free mice. Also, pretreatment with sorafenib (30mg·kg-1day-1and60mg·kg-1day-1,2weeks) caused a substantial reduction of NK-mediated cytotoxicity against YAC-1cells in both tumor-bearing and tumor-free mice. Notably, depletion of NK cells with the neutralized antibody of NK1.1significantly promoted the formation of lung metastatic foci compared to the NK-normal C57BL/6mice, but it eliminated the prometastatic effect of sorafenib pretreatment in NK-depleted C57BL/6mice.3. Sorafenib inhibited the proliferation and cytotoxicity of NK cells mainly by blocking PI3K/AKT and Raf/MEK/ERK signaling pathwayTo verify the underlying mechanism of suppression of NK cells induced by sorafenib, we isolated NK cells from healthy donors and C57BL/6mice to study the function and signaling pathways and cultured human NK cell line NK-92MI to examine the proliferation of NK cells. The rate of inhibition of NK-92MI cell proliferation depended on the concentration of sorafenib:more than60%at5μM for72h, and almost100%at10μM. We found that30μM PI3K-specific inhibitor LY294002inhibited growth in a similar fashion as10μM sorafenib, indicating that sorafenib may affect the PI3K/AKT pathway in NK92-MI. Like sorafenib, LY294002(10μM,20μM, and30μM) had a dose-dependent effect on NK-92MI growth. Akt phosphorylation was inhibited in a dose-and time-dependent manner by sorafenib or LY294002. Sorafenib treatment caused substantial reduction of NK-mediated cytotoxicity against K562cells. In addition, sorafenib also dose-dependently impaired IFN-y production in NK cells cocultured with K562cells. Furthermore, presence of K562cells induced expression of CD107a in NK cells, a marker of activated NK cells, and sorafenib significantly reduced its expression in a dose-dependent manner. In line with the results obtained in analysis of cytotoxicity and degranulation, sorafenib treatment also drastically altered the expression of stimulatory receptor NKG2D. Addition of the specific MEK inhibitors PD98059and U0126,20μM, was,followed by cytotoxicity and IFN-y production similar to that observed with10μM sorafenib. The effects of sorafenib on NK cell activation may be due to inhibition of signaling events earlier than1h and survival by the inhibition of signaling events later than12h.4. Herbal extract "songyou yin"enhances the antitumor effect of sorafenib through modulating natural killer cells activity in xenograft model of hepatocellular carcinomaThe purpose of this part is to examine whether the herbal exact "songyou yin"(SYY-comprises5herbs)) enhance the antitumor effect of sorafenib against hepatocellular carcinoma (HCC) when used in combination and investigate the underlying mechanisms. In LM3-RFP orthotopic nude mouse models, sorafenib (30mg/kg/d) significantly reduced the tumor volume and reduced the number of pulmonary metastatic tumor foci compared to control. However, when the number of metastatic tumor foci was standardized by the liver tumor size (standardized number of lung metastases, SNLM), sorafenib significantly increased the SNLM compared with control. SYY alone at a low dose did not inhibit tumor growth and metastasis but did exert a synergistic inhibitory effect on tumor growth and metastases when combined with sorafenib in vivo. Analysis of survival of mice showed pronounced prolongation of median survival by the combination therapy in comparison with sorafenib alone. To confirm this effect, we reconducted the combined therapy in HepG2-GFP (low metastatic potential) and the results were just similar to that in LM3-RFP nude mice models.To elucidate the underlying mechanism by which the combination of sorafenib and SYY exert enhanced anticancer activity in vivo, we tested the direct cytotoxicity of soafenib and SYY on LM3and HepG2cells in vitro. There was no obvious additive antitumor effect on LM3and HepG2cells when sorafenib was combined with SYY. Since sorafenib has been found to inhibit the reactivity of NK cell against tumor cell both in vitro and in vivo, we next evaluated whether the modulation of NK cells by SYY mediate the increased antitumor activity of combination therapy. In Hepal-6orthotopic C57BL/6models, SYY indirectly protect NK cells from impairment by sorafenib in vivo to some extent. Moreover, in pretreated experimental lung metastasis models, much less pulmonary metastatic foci were found in combination pretreated mice than that of sorafenib pretreated mice. Although SYY only marginally enhanced the activity of NK cells and the difference was statistically insignificant compared with control, we found NK cells in the presence of plasma collected from SYY treatedLM3-R model exhibited an enhanced cytotoxicity against YAC-1cells. Levels of IFN-γ and MCP-1were elevated significantly in the combination of sorafenib and SYY treated mice compared to that of sorafenib treated mice, suggestive of indirect immune-potentiating activity of NK cells by SYY that contributed to its increased antitumor efficacy when combined with sorafenib.Conclusions1. Sorafenib pretreatment promoted metastatic potential of HCC in mice2. Sorafenib inhibited NK cells rendering the host more susceptive to tumor growth and metastasis. Sorafenib could inhibit the proliferation of NK cells and impair their reactivity by affecting phosphorylation of AKT and ERK1/2, respectively.3. The Chinese herbal "SYY" at a low dose could further inhibit tumor progression and prolong host survival when combined with sorafenib by indirect immune-potentiating activity of NK cells, which is promising to be combined with sorafenib for HCC patients.The potential application of this work1. Sorafenib exhibited direct inhibitory effects on NK cells, suggestive of the necessity of surveillance about antitumor immunity for the patients with advanced HCC who received sorafenib treatment.2. These results suggested the Chinese herbal "SYY" could protect NK cells from impairment induced by sorafenib to some extent, and other immunotherapeutic approaches to activate NK cells may potentially enhance the therapeutic efficacy of sorafenib in HCC patients.The novelties of this work:1. The present studies demonstrated that the off-target immunosuppressive property of sorafenib was mediated by suppression of NK cells, which may account for the modest therapeutic efficacy of sorafenib for the advanced HCC patients.2. The Chinese herbal "SYY" attenuated the suppression of natural killer(NK) cells induced by sorafenib and enhanced the antitumor effect of sorafenib in vivo, suggested that the combination of sorafenib and SYY may be an effective regimen for its potential clinical applications.