NB potentiates cytotoxicity of DOX against A549 cells through enhancing cellular uptake
Firstly, the acute toxicity of borneol was examined. As illustrated in Table. s1, the median lethal dose (LD50) of synthetic borneol (SB) was 3129 mg/kg and the 95% confidence interval at 1750 ~ 5000 mg/kg, while 5000 mg/kg and 2016 ~ 9810 mg/kg were found in NB, respectively. Moreover, significant pathology changes were found in liver, spleen and lung only in SB (5000 mg/kg) treatment mice, while slightly inflammation effects were found in stomach and intestinum tenue in both SB and NB (5000 mg/kg) treated mice (Fig. s1). These results suggest that NB exhibits higher safety index than SB, thus NB was selected for chemo-sensitizing study. As illustrated in Table s2, NB was able to enhance the cytotoxic effects of DOX against A549 cells with the IC50 value of DOX decreased to 0.18 ± 0.06 µM. Similar enhancive effects of NB on 5-FU, paclitaxel and cisplatin were also observed. These results suggest the sensitizing capacity of NB on chemotherapeutic agents against A549 cells, and we selected NB and DOX for further study. As shown in Fig. 1a, DOX treatment alone slightly restrained the A549 cells growth at the concentration range from 0.06 µM to 0.25 µM. However, strongly cytotoxicity effects were observed when cells pretreated with NB for 12 h followed by low dose of DOX for 72 h with suppression ratio increased up to 40.26%. The long term clonogenic assay also confirmed these results (Fig. 1b and 1c). Furthermore, we also explore the cytotoxicity of NB and DOX towards normal colonic NCM-460 cells. We found that NCM-460 cells were less inhibited as compared to A549 cells, which indicated that NB potentiated DOX induced prominent cells survival reduction mainly in tumor cells (Fig. 1d).
In order to investigate the underlying mechanism of the cytotoxicity effects induced by NB and DOX, the intracellular accumulation of DOX was examined. As illustrated in Fig. 1g, NB pretreatment dramatically augment the intracellular accumulation of DOX when compared to DOX treatment alone as evidenced by more remarkable red fluorescence, which is in accordance with the FACS data in Fig. 1e and 1 f. Moreover, drug resistance mediated by P-glycoprotein (P-gp) also confirmed to be one of the major causes of DOX-resistance in NSCLC[31]. Therefore, P-gp functional analysis was also conducted. As shown in Fig. 1g and 1 h, NB was able to restrain the P-gp action in a time and dose-dependent manner as indicated by the increased intracellular fluorescence intensity of rhodamine 123. Together, these results demonstrate that NB inhibits P-gp function to potentiates the cytotoxic effects of DOX against A549 cells.
NB synergizes with DOX to induce apoptosis through activating caspases cascade in A549 cells
We next employed Flow cytometry assay to examine the mode of cell death triggered by this combination treatment. As depicted in Fig. 2a and 2b, no significant cycle distribution changes were observed in NB treatment groups, while significant accumulated population of Sub-G1 (17.05%) and G0/G1 phase were observed after DOX incubation, which suggests that DOX is able to trigger both cell cycle arrest and apoptosis in A549 cells. However, significant apoptosis effects were observed in cells that pretreated with NB followed by DOX, as illustrated by the increased population of Annexin V and PI double positive cells in Fig. 2c. For instance, 6.16% apoptosis cells were observed in NB treatment alone, while 15.7% was found in DOX incubation groups. However, NB pretreatment significantly enhanced the killing potency of DOX with the apoptosis proportion increased to 41.5%, which is further confirmed by the morphology changes of treated cells (Fig. 2d). These results indicate that NB sensitizes A549 cells to DOX-mediated killing potency by inducing apoptosis.
In order to further confirm the apoptosis inducing effects of NB combined with DOX, fluorometric assay to examine caspases activation was examined. As demonstrated in Fig. 2e and 2f, both NB and DOX were able to induce slight activation of caspase-3/-8/-9. However, cells pretreated with NB followed by the incubation of DOX were found to synergistically increased the activation of caspase-3/-8/-9, which indicates the activation of extrinsic and intrinsic apoptosis pathways. The western blotting assay results of significant cleavage of caspase-9, caspase-8, caspase-3, and PARP further strengthens these findings. Together, these results demonstrate that NB enhances DOX-induced A549 cells death mainly by inducing apoptosis through activating extrinsic and intrinsic apoptosis pathways.
Activation of ROS-mediated pathway contributes to the apoptosis effects induced by NB and DOX
Extrinsic and intrinsic apoptosis signal will converge to mitochondria. Therefore, MitoTracker Green probe was used to examine the integrity of mitochondria. The results show that compared to the extensively interconnected and filamentous appearing network of mitochondria observed in the control treatment, NB slightly triggered mitochondria structure damage after 8 h incubation. Meanwhile, slightly destroyed structure can be observed after cells treated with DOX alone for 4 h. However, small fragmentation of mitochondria was observed early at 2 h after the combined incubation of NB and DOX, and followed by notable damage at 4 h, and became progressively worse at 8 h (Fig. 3a). The depleted mitochondrial membrane potential (∆ψm) also confirmed the damage of mitochondria (Fig. s2). Furthermore, we found that Bcl-2 expression was strongly inhibited by the combined treatment of NB and DOX, while the expression of tBid and Bax was significantly upregulated (Fig. 3b). These results suggest that NB synergized the killing potency of DOX is highly correlated with mitochondria dysfunction.
Triggering DNA damage has been found to be one of the major mechanisms of DOX[32], which may regulate mitochondria-mediated apoptosis through activating p53. In this study, we found that NB significantly enhanced DOX-induced DNA damage, as evidenced by the upregulated protein expression level of phosphorylated ATM, ATR and histone (Ser 139), as well as phosphorylated p53 (Ser 15) (Fig. 3c). These results indicate that DNA damage-mediated p53 activation contributes to the sensitizing effects of NB on DOX in A549 cells.
Mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways paly important role in the proliferation and metastasis of cancer cells. We next evaluate whether the combination treatment of NB and DOX could affect the function of these kinases. As depicted in Fig. 3d and 3e, NB and DOX combined treatment dramatically suppressed phosphorylated-AKT and phosphorylated-ERK while upregulated the phosphorylation of p38 MAPK (Thr180/Tyr182) and SAPK/JNK (Thr183/Tyr185). These results suggest that NB synergizes with DOX induces A549 cells apoptosis through inhibiting AKT and ERK activation, but stimulating p38MAPK and JNK activation.
Expanding evidences have disclosed the pivotal role of ROS in the anticancer mechanisms of DOX[33]. And ROS exhibits regulation role in the modulation of Mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways[34]. As shown in Fig. 3f, both NB and DOX were able to trigger ROS generation, however, strongly upregulation of ROS accumulation was achieved when cells incubated with the combined treatment of NB and DOX. Moreover, N-acetyl-L-cysteine (NAC), a thiol reducing antioxidant, pretreatment significantly inhibited cells growth suppression and cell apoptosis induced by the combined incubation of NB and DOX (Fig. 3g and 3 h), which suggested the upstream role of ROS. Taken together, these results demonstrate that NB dramatically augments DOX-triggered cells apoptosis is relied on activation of ROS-mediated DNA pathway.
NB enhances the chemosensitivity of A549 cells to DOX through targeting TRPM8
SPR biosensor technology is widely used for peptide screening and small molecule drugs screening[35]. Since NB enhances the anticancer activities of DOX, MS-SPRi technology was exploited to elucidate the candidate targets of NB (Fig. 4a). The MS score is used to evaluate the binding strength of NB on proteins, and Peptide Spectrum Matches (PSMs) is represented as the number of peptide map matches. As shown in Fig. 4b and 4c, 31 cell lysis proteins of A549 cells were captured by NB. From Gene Ontology cluster analysis in Biological Process, we found that most of these captured proteins are involved in cellular process regulation (Fig. 4d). Additionally, 19 of 31 proteins are found to take part in the regulation of stress response. And 21% of these 19 proteins are belong to ion channel proteins, such as GABRA5, TRPM8, TRPA1 and TRPV3 (Fig. 4e and 4f). The MS score of GABRA5 (PSMs = 66) is found to be highest and followed by TRPM8 (PSMs = 31). Since GABRA5 mainly participates in neuron-neuron synaptic transmission regulation [36]. We speculate that TRPM8 may be an important target of NB, and TRPM8 was selected for further study.
TRPM8 has been considered as a cancer marker of lung cancer[37]. Lung adenocarcinoma is found to overexpress of TRPM8, and its expression is negatively correlated with the survival rate of patients according to TCGA and PROGgene database (Fig. 4g and 4 h)[38, 39], which suggests that targeting TRPM8 may help to improve the therapeutic index of lung cancer treatment. TRPM8 is an important calcium channel protein[40]. Therefore, we first detected whether NB could affect intracellular dissociative calcium concentration in A549 cells. As shown in Fig. 4i, the intracellular fluorescence signal of A549 cells loaded with Fluo-3AM probe was enhanced after NB addition and the signal was upregulated in according with the increased concentration of NB, indicating the capacity of NB in inducing calcium mobilization. In order to further investigate whether NB affects calcium accumulation through interacting with TRPM8, short interference double-stranded RNA (siRNAs) of TRPM8 interference assay was employed. As shown in Fig. 4j and 4 k, the protein expression level of TRPM8 was dramatically inhibited after 48 h transfection. The intracellular calcium signal induced by NB was significantly downregulated in the TRPM8 knock down cells when compared with siNC control groups (Fig. 4l). These results further indicate the important role of TRPM8 in calcium immobilization triggered by NB stimulation. Furthermore, we also found that knock down of TRPM8 restrained the accumulation of intracellular ROS (Fig. 4m and 4n) induced by NB combined with DOX and reversed the killing capacity of DOX against A549 cells sensitized by NB (Fig. 4o).
Except for TRPM8, we found that the MS score of androgen receptor (AR) was 1077.15 and the PSMs was 93, which indicated that AR may also participate in the sensitizing effects of NB. Furthermore, patients with high expression level of AR is also found to possess lower survival rate[39]. Therefore, in order to evaluate whether the synergistic effects of NB and DOX was relied on the interaction of NB on AR, AR knock down assay was also employed. As shown in Fig. s3, no significant changes of anticancer effects triggered by NB and DOX were observed between siAR knock down groups and siNC control groups. This suggests that AR may not participate in the regulation of NB as a DOX sensitizer. Taken together, these results suggest that NB may synergize with DOX to achieve potent anticancer efficiency through triggering intracellular Ca2+ mobilization by interacting with TRPM8.
NB augments the antitumor activities of DOX in vivo.
To further evaluate the potential therapeutic efficacy of NB and DOX, we exploited xenografts nude mice model assay. As shown in Fig. 5a-5c, both borneol and DOX were found to inhibit tumor growth. After 15 days treatment, the tumor volume decreased to 268.45 mm3 ± 17.01, and the tumor inhibition rate was 39.79%. However, the tumor volume was significantly inhibited in the combined treatment of NB and DOX. For example, the average tumor volume of p.o. NB + i.v. DOX group and i.v. NB + i.v. DOX group was 205.61 mm3 ± 17.05 and 164.42 mm3 ± 25.03, respectively. The tumor inhibition rate was 53.95% and 63.16% (Fig. 5d). In addition, we found that intravenous injection of borneol and DOX exhibited more potent anti-tumor effect than oral administration schedule, which may be due to more potent blood bioavailability of NB after intravenous administration[27], which promoting the accumulation of DOX in the tumor site and inducing more significant anti-tumor effects. The accumulation of DOX in tumor tissues also confirmed this result. For instance, the content of DOX was 1.89 (µg/g tumor tissue) ± 0.05 in DOX treatment groups and 3.34 (µg/g tumor tissue) ± 0.69 in p.o. NB + i.v. DOX groups. However, DOX concentration in the tumor tissues of i.v. NB + i.v. DOX groups was much higher with the content at 5.27 (µg/g tumor tissue) ± 0.41 (Fig. 5e). CD34 and Ki67 expression in tumor sections also verified these findings (Fig. 5g). Moreover, no significant pathological changes in the major organs of heart, liver, spleen, lung and kidney and TG, UA, ALB, TP, LDH were found between the combined treatments groups and control groups (Fig. 5h and 5i). Taken together, these results demonstrate that NB and DOX synergized to suppress tumor growth in vivo.