Positive SP/NK-1R expression in iCCA patients and induction of tumour progression by targeting NK-1R in human iCCA cells
To investigate the expression of substance P (SP) and neurokinin-1 receptor (NK-1R) in intrahepatic cholangiocarcinoma (iCCA), with the approval and supervision of the ethics committees, we collected tumor samples of patients with iCCA who underwent surgical resection in the Second Affiliated Hospital of Nanjing Medical University. We found that SP/NK-1R was significantly expressed in the tumor samples of iCCA (Fig. 1A, B). Additionally, we explored the expression of NK-1R in RBE and HCCC-9810, by immunofluorescence staining and western blotting. The results showed that NK-1R was markedly expressed in iCCA cells (Fig. 1C, D).
It is well known that SP can promote mitosis in a variety of tumor cells[30]. We cultured RBE and HCCC-9810 for 48 h with SP at the concentrations of 1 nM, 10 nM, 100 nM and 1000 nM, and the MTT assay showed that SP could significantly enhance cell viability in a concentration-dependent manner (Fig. 1E). After treatment with 100 nM of SP, iCCA cells exhibited an enhanced proliferative capacity (Fig. 1F, G). These results showed that SP/NK-1R was notably expressed in iCCA, and the activation of SP/NK-1R signaling pathway could promote cell proliferation of iCCA.
Blocking NK-1R by aprepitant inhibiting proliferation of iCCA cells
It has been indicated that NK-1R antagonist, aprepitant can effectively inhibit proliferation and growth of several kinds of tumors. In order to examine the anti-proliferative effects of aprepitant on iCCA cells, we cultured RBE and HCCC-9810 with aprepitant by concentration gradients (0 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM) and temporal gradients (24, 48 and 72 h), respectively. Aprepitant demonstrated remarkable potency against cell proliferation of RBE and HCCC-9810 with IC50 of 40 μM and 12 μM, respectively.
After administration of aprepitant (RBE: 20 μM, 40 μM, 80 μM; HCCC-9810: 6 μM, 12 μM, 24 μM) for 2 h, the proportion of EdU incorporated into nuclear DNA decreased significantly in a dose-dependent manner (Fig. 2B). Additionally, iCCA cells could hardly form colonies (Fig. 2C). The above results suggested that aprepitant effectively weakened cell proliferation and colony-forming ability of iCCA cells.
Aprepitant inhibiting human iCCA xenograft growth
RBE and HCCC-9810 cell suspensions were subcutaneously injected into the right axilla of BALB/c nude mice. When the nodules were palpable at the injection site, the mice were randomly divided into two groups. The control group received intraperitoneal injection of 0.9% saline and another group received intraperitoneal injection of aprepitant (10 mg/kg). Compared with control group, administration of aprepitant remarkably reduced the volume and weight of xenograft tumors without affecting the body weight of nude mice, which revealed that aprepitant exhibited anti-iCCA potential in vivo (Fig. 3A-D).
Aprepitant inducing mitochondria-dependent apoptosis through JNK/MAPK pathway
In order to explore whether cell apoptosis is involved in the cytotoxic effects of aprepitant on iCCA, we incubated the cells with different concentrations of aprepitant for 24h. The apoptotic rate of iCCA cells increased significantly after treatment with aprepitant (Fig. 4A, RBE: 20 μM 24.78%, 40 μM 33.99%; HCCC-9810: 6 μM 24.03%; 12 μM 38.28%). Furthermore, the Bcl-2/Bax ratio decreased and the expression of cleaved caspase-3, cleaved caspase-9 and cleaved PARP increased in a dose-dependent manner (Fig. 4B).
Recent studies suggest that aprepitant can accelerate endoplasmic reticulum calcium overload and mitochondrial production of reactive oxygen species (ROS) to induce apoptosis[31]. High concentrations of intracellular calcium and severe oxidative stress are recognized as triggers of the intrinsic pathway of apoptosis, and the depolarization of mitochondrial membrane potential (MMP) is considered to be an linchpin of mitochondrial pathway[32]. We observed significant accumulation of intracellular ROS 15 min after treatment with aprepitant in a dose-dependent manner (Fig. 4C). Besides, JC-1 probe was used to detect the changes of MMP after administration of aprepitant. The red fluorescence intensity of mitochondria decreased and the green fluorescence intensity increased significantly in RBE and HCCC-9810, suggesting the depolarization of MMP (Fig. 4D). Thus, aprepitant can exert a cytotoxic effect on iCCA by mediating apoptosis in mitochondrial pathway.
MAPK signaling is a major pathway downstream of NK-1R activation[33]. Western blotting results showed that aprepitant significantly increased phosphorylation of ERK, JNK and p38, but downregulated the expression of phospho-AKT in a dose-dependent manner (Fig. 5A). In order to determine which kinases in MAPK pathway play a decisive role, before administration of aprepitant, we pretreated iCCA cells with trametinib (ERK pathway inhibitor, 2 nM), SP600125 (JNK pathway inhibitor, 100 nM) and SB202190 (p38 pathway inhibitor, 50 nM) for 2 h, and then detected the changes of apoptotic rate by flow cytometry. The results demonstrated that SP600125 significantly reversed the increase of apoptotic rate induced by aprepitant, while neither trametinib nor SB202190 could (figure 5B). Taken together, aprepitant could induce cell apoptosis of iCCA by activating JNK MAPK pathway.
Aprepitant inducing autophagy of iCCA cells
To determine whether pro-apoptotic effect is the only cytotoxic effect of aprepitant, we pretreated iCCA cells with z-VAD-fmk (pan-caspase inhibitor, 40 μM) for 1 h, and then incubated the cells with aprepitant for 24 h. The results of MTT showed that z-VAD-fmk could not restore the cell viability to the level of the control group (Fig. 6A). Nevertheless, after pretreatment with ROS scavenger, NAC (3mM) for 2 h, the cell viability was almost completely restored (Fig. 6B). These findings indicated that besides the pro-apoptotic effect, there were other mechanisms involved in the cytotoxic effect of aprepitant on iCCA, and the accumulation of ROS remained the upstream signal of other mechanisms.
As accumulation of ROS, which is mainly released by mitochondria, plays an important role in activating autophagy[34], western blotting was performed to investigate whether aprepitant could facilitate cell autophagy of iCCA. We found that treatment with aprepitant significantly resulted in an increase of LC3II/LC3I and decrease of SQSTM1/p62, while aprepitant had no significant effect on the expression of ATG7 and Beclin-1 (Fig. 6C). In addition, we observed a significant increase in the number of autophagosome by electron microscope (Fig. 6D).
To further clarify the dynamic process of autophagy, we transfected iCCA cells with mRFP-eGFP-LC3 adenovirus, and mRFP and eGFP were used to label and track LC3. After treatment with aprepitant, the formation of yellow fluorescent mRFP+/eGFP+ (autophagosome) was significantly increased, and subsequently, the intensity of red fluorescent mRFP+/eGFP- (autolysosome) increased gradually, suggesting that aprepitant promoted the formation of autophagosome and autolysosome in iCCA (Fig. 6E). Moreover, rapamycin (20 μM) promoted the red fluorescence accumulation induced by aprepitant, and 3-MA (5 mM) reduced the red fluorescence accumulation, while CQ (25 μM) promoted the yellow fluorescence accumulation (Fig. S1). In summary, these results indicated that aprepitant could induce cell autophagy of iCCA.
ROS accumulation regulating aprepitant-induced downstream signaling pathways, apoptosis and autophagy
We pretreated RBE and HCCC-9810 with ROS scavenger, NAC (3 mM) for 2 h. Flow cytometric analysis showed that NAC could rescue cell apoptosis induced by aprepitant (Fig. 7A). NAC could reverse the aprepitant-induced increase of Bax, cleaved caspase-3, cleaved caspase-9, cleaved PARP and LC3II/LC3I and the decrease of p62 (Fig. 7B). Furthermore, NAC significantly inhibited the phosphorylation level of ERK, JNK and p38 induced by aprepitant, but had no significant effect on phospho-AKT (Fig. 7C). The results further confirmed that the accumulation of ROS played a key role in regulating MAPK pathways, cell apoptosis and autophagy induced by aprepitant.
Aprepitant inducing crosstalk between apoptosis and autophagy
Autophagy and apoptosis may be initiated by common upstream signal molecules, such as the transcription factor p53 and BH3-only protein family, and in some cases, cell life activities can be switched between these two processes in multiple ways[35]. To explore the crosstalk between apoptosis and autophagy of iCCA cells mediated by aprepitant.
We pretreated the cells with z-VAD-fmk (40 μM), 3-MA (5 mM) and the joint treatment of these two. Both z-VAD-fmk and 3-MA pretreatment could partially recover the decrease of cell viability induced by aprepitant, while the joint treatment could almost completely recover (Fig. 8A). Z-VAD-fmk pretreatment can further promote the accumulation of red fluorescence (mRFP+/eGFP-) in eGFP-mRFP-LC3-transfected cells (Fig. 8B). LC3II/LC3I was further increased after z-VAD-fmk pretreatment, while cleaved PARP and cleaved caspase-9 were further decreased after 3-MA pretreatment (Fig. 8C-D). Compared with the control group, the level of LC3II/LC3I increased slightly in z-VAD-fmk group, but there was no significant change in the expression of cleaved caspase-9 and cleaved PARP in 3-MA group (Fig. 8C-D). Rapamycin (20 μM) pretreatment significantly facilitated the aprepitant-induced apoptosis, while 3-MA (5 mM) pretreatment showed a completely opposite effect (Fig. 8E-F).