Rabbit anti-caspase-3 and anti-PARP antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The rabbit anti-LC3B antibody was obtained from Sigma (St. Louis, MO, USA). The mouse anti-cathepsin D antibody was obtained from Huada Biotechnology Co. (Beijing, China). The mouse anti-β-actin antibody was obtained from ZSGB-BIO (Beijing, China). CQ and 3-methyladenine (3-MA) were purchased from Sigma (Deisenhofen, Germany), prepared initially as a 20 mM stock solution by dissolving in physiological saline (Minkang, China) and dissolved in RPMI-1640 medium to the desired concentration before the experiment, respectively. HNK was purchased from Shanghai Ziyi Reagent Factory (Shanghai, China) and rapamycin was purchased from Shanghai Qianchen Reagent Co. (Shanghai, China). The two agents were dissolved in dimethyl sulfoxide (DMSO) as a 20 mM and 218 µM stock, respectively. Before use in the experiments, each stock solution was diluted with RPMI-1640 medium to the desired final concentration. The caspase-3 inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) was purchased from Beyotime (Jiangsu, China) and acridine orange was purchased from Sigma.

The human NSCLC cell lines A549 and H460 were used in the experiments and stored in our laboratory. All the cell lines were cultured in RPMI-1640 medium (Thermo Scientific) supplemented with 10% fetal calf serum (Gibco), 1% penicillin/streptomycin (North China Pharmaceutical, China) and maintained in a 5% CO₂ incubator at 37°C.

Cells were seeded in 96-well microtiter plates and exposed to different concentrations of the chemicals overnight for 48 h. Then, the sulforhodamine B (SRB) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability. The SRB assay is briefly described as follows. After the treatment period, the cells were fixed with cold 10% (w/v) trichloroacetic acid and incubated for 60 min at 4°C. After the supernatant was discarded, double distilled water was used to wash the plates five times and air dried. Then, SRB solution [0.4% (w/v) in 1% acetic acid)] was applied to stain the cells for 5 min at room temperature. After staining, the plates were washed with 1% acetic acid five times to remove the unbound dye and air dried. SRB was subsequently solubilized with 10 mM Tris (pH 10.5), and the absorbance was read at 570 nM on a microplate reader (Multiskan MK3; Thermo Scientific). The MTT assay was carried out as previously described (14).

The coefficient of drug interaction (CDI) was applied to evaluate the effect of drug interaction. CDI was calculated as follows: CDI = AB / ( A × B )

Where AB is the survival rate of the cells affected by the combined drugs. A or B is the survival rate of cells affected by each drug alone. When CDI is <1, there exists a synergistic effect of the two drugs (15).

Cells were plated into 6-well plates and were treated with different concentrations of the agents for 20 h. Then, cell apoptosis was examined by the Annexin V-FITC/PI apoptosis detection kit (Biosea Biotechnology, Beijing, China) according to the manufacturer's instructions. Briefly, the cells were collected, fixed and stained with 10 µl of Annexin V-FITC and 5 µl of PI. Flow cytometric analysis was performed on 1×10⁴ cells/sample by a FACS Coulter EPICS XL in less than 1 h and analyzed with a FACSCalibur using Summit software (both from Beckman Coulter, Inc., Fullerton, CA, USA). Caspase activation, an additional indicator of apoptosis, was also detected by western blotting as described below.

Cells were exposed to the chemicals for different times and then harvested. The cells were lysed and analyzed by western blotting as previously described (16).

The protocols of animal experiments were, according to the rules of Good Laboratory Practice for Non-Clinical Laboratory Studies of Drugs issued by the National scientific and technologic Committee of P.R. China, approved by the Experimental Animal Center of the Institute of Medicinal Biotechnology.

We obtained 6-to 8-week-old female BALB/c nude mice (nu/nu) from Vital River Co. (Beijing, China) and housed them under pathogen-free conditions with laboratory chow and water. H460 cells (5×10⁶/mouse) were injected into the right armpits of the nude mice. After 3 weeks, tumors resulting from donor animals were aseptically dissected and cut into 2×2×2 mm³ pieces. The pieces were then transplanted into fresh mice using trocar needles. Prior to the tumors reaching ~100 mm³, the mice were randomized into 5 groups (6/group) based on body weight and tumor volume. The mice were administered HNK (50 mg/kg/day, 5 times/week, i.p.) and CQ (100 mg/kg/day, 5 times/week, p.o.). Control nude mice were injected with saline or 20% intralipid (HNK dissolved in 20% intralipid) (17). The long and short diameters of the tumors (a and b, respectively) were measured using a caliper twice a week and the body weights were recorded at the same time. The tumor volume was calculated according to the formula, V = ab²/2. The animal experiment lasted for 21 days. At the end, the tumor tissues were used to assess the LC3-II expression with western blotting.

The abundance of acidic lysosomes in the cells was evaluated with AO staining. After treatment with HNK for 24 h, the cells were stained with acridine orange (1 µg/ml) for 15 min at 37°C in the dark and then washed with PBS. The images were captured using a fluorescence microscope (Vert. A1; Zeiss, Germany) using a 490-nm band-pass excitation filters and a 515-nm long-pass barrier filter. Based on the acidity, the lysosomes appeared as orange fluorescent cytoplasmic vesicles (18).

Experimentally siRNA targeting human cathepsin D (cathepsin D-siRNA) was purchased from Ruibo Biotechnology (Guangzhou, China), and the following siRNA sequence was CCUCGUUUGACAUCCACUAdTdT (19). The cells were transfected with siRNAs using the Ruibo FECT™ CP transfection kit according to the manufacturer's instructions. Briefly, the cells were plated in a 6-well plate for an overnight incubation, and then the medium was altered to antibiotic-free for transfection. After the transfected solution in an antibiotic-free condition was added to the plates, the cells were incubated at 37°C for 24 h and treated with HNK for another 24 h. Finally, the efficiency of the RNAi was measured by western blotting.

All of the experiments were conducted independently at least three times. All the data are represented as mean ± SD.

In order to investigate the antitumor effect of combining HNK with CQ, an SRB assay was used to validate the cytotoxicity of HNK or CQ alone or their combination. As shown in Fig. 2A, HNK or CQ decreased the viability of the A549 and H460 cells in a dose-dependent manner at 48 h.

Then the antitumor effect of the combination of HNK and CQ was investigated via the SRB assay. The cells were treated with CQ at the doses of 10 and 20 µM, and then doses of HNK were added at the same time. CQ sensitized the H460 and A549 cells to HNK (Fig. 2B) and the majority of the CDIs in the combination doses were <1 (Fig. 2C). Therefore, there was a synergistic effect between CQ and HNK.

To determine the effect of the two drugs on the apoptosis of human NSCLC cells, both cell lines were treated with HNK (40 µM for the A549 and 30 µM for the H460 cells) or CQ (20 µM) alone or in combination for 24 h. Annexin V-FITC and PI staining indicated early and late apoptotic and necrotic cells, respectively. The apoptotic cells were increased in the CQ+HNK group compared with the CQ or HNK group. The apoptotic percentages of the A549 cells were 2.53, 3.77 and 6.42% for CQ, HNK and CQ+HNK, respectively, and the apoptotic percentages of the H460 cells were 6.65, 6.96 and 14.25%, respectively (Fig. 3A and B).

After the combination of CQ and HNK induced NSCLC cells to undergo apoptosis, western blotting was applied to investigate the levels of marker protein expression in the apoptotic pathway. The CQ+HNK group exhibited decreased PARP and increased cleavage after the two drugs were combined as compared to CQ or HNK alone (Fig. 3C).

Finally, zVAD.fmk, a broad-range caspase inhibitor, was used to examine whether the cell death induced by the combination of HNK and CQ was caspase-dependent. zVAd.fmk significantly decreased the HNK and CQ-triggered increased levels of cleaved caspase-3 and cleaved-PARP in the A549 and H460 cells (Fig. 3D).

The above results demonstrated an enhanced effect on the proliferation and apoptosis of NSCLC cells following the combination of HNK and CQ. Thus, we explored whether CQ can augment the antitumor effect of HNK in vivo. According to the method as previously described, administration of the drugs was started at day 7 after tumor blocks were implanted in the BALB/c nude mice. No incidence of death in the experimental groups occurred during the experiment. Fig. 4A shows that the average body weight in the groups did not significantly change and was stable. Thus, the doses of CQ and HNK were tolerated. The volume of the mouse tumors was reduced in the CQ, HNK and CQ+HNK groups as compared to the controls and the percentage of tumor growth inhibition in the three groups on day 21 was 38.81, 64.92 and 76.91%, respectively (Fig. 4A). Thus, the reduction in tumor volume in the CQ+HNK group was more significant than that in the CQ or HNK group.

It has been reported that HNK produces pharmacological functions via influencing autophagy, and CQ is used as an autophagic inhibitor during autophagy studies. Thus, we further investigated the LC3-II expression in tumor tissues from the H460 xenografts in BALB/c nude mice. In the experiment, the tissues from three mice in each group were used to conduct western blotting. The results revealed that LC3-II expression was increased after CQ, HNK or CQ+HNK treatment. The average intensity of the LC3-II band in the CQ+HNK group was greater than that in the CQ or HNK group (Fig. 4B).

From the above results, we found that HNK increased the expression of LC3-III similar to CQ. To further show the role of autophagy in the antitumor effects of HNK or coupled with CQ in NSCLC cells, the expression levels of p62 and LC3-II in the A549 and H460 cells were examined after HNK treatment. As shown in Fig. 5A, HNK at a concentration as low as 10 µM was able to increase the p62 and LC3-II expression in the A549 cells, and it increased levels of the two proteins at the concentration of 20 µM in the H460 cells. In addition, HNK increased the levels of LC3-II in the A549 and H460 cells, which occurred at 8 h and were maintained at least for 24 h. The p62 expression level was significantly increased at 16 h and was maintained up to 24 h (Fig. 5B). From this, we found that prolonged treatment of HNK generated cell line-dependent results on p62 and LC3-II regulation.

Autophagy is a lysosome-dependent membrane degradative process, thus the lysosome stability was examined by AO staining. During the experiment, the nucleus was stained green and acidic vesicles (lysosomes and autophagolysosomes) were present in red fluorescence. As shown in Fig. 5C, the red fluorescence intensity was increased in the cytoplasm of both cell lines as compared to that in the control group after HNK treatment for 24 h.

In order to investigate the role of autophagy in the antitumor effect of HNK or combined with CQ, various inhibitors were applied as tools to inhibit autophagy. In this experiment, CQ and 3-MA, as autophagy inhibitors at different stages, were used to examine the role of autophagy on the cytotoxicity induced by HNK. The lysosome-inhibitor CQ inhibited the fusion of lysosomes and autophagosomes, causing the accumulation of LC3. 3-Methyladenine (3-MA) inhibited the initiation of autophagy (20). At first, the cell viability was measured by MTT assay. HNK, CQ or 3-MA was able to suppress the proliferation of the A549 and H460 cells. The combination of HNK and CQ decreased the viability of both cell lines more potently than HNK or CQ alone, but there was no enhanced effect on the inhibition of proliferation by HNK or CQ combined with 3-MA, respectively (Fig. 6A).

Secondly, we conducted western blotting to detect the expression of p62 and LC3-II after HNK, CQ, 3-MA or the combinations. The results demonstrated that HNK, CQ or 3-MA all induced the expression of p62 in the A549 and H460 cells, but HNK or CQ alone increased the level of LC3-II expression. HNK or CQ alone augmented the level of p62 induced by 3-MA and increased the LC3-II expression level even though 3-MA did not induce the LC3-II expression. However, the combination of HNK and CQ did not clearly change the level of the two autophagic markers as compared to HNK or CQ alone (Fig. 6B). HNK inhibited the fusion of lysosomes and autophagosomes and possessed various characteristics of an autophagy inhibitor such as CQ.

From the above results, HNK inhibited autophagy during our experiments. Thus, rapamycin and CQ, which induces and inhibits autophagy respectively, were used to verify the role of autophagy in the cell death of human NSCLC cells induced by HNK. As shown in Fig. 7A, rapamycin did not enhance the anticancer effect of HNK at the low concentrations of 10 and 20 µM or CQ in both cell lines, but an enhanced effect of CQ and HNK was noted. The western blot results (Fig. 7B) showed that rapamycin reduced p62 expression and increased LC3 expression in the A549 and H460 cells when in combination with CQ or HNK. Surprisingly, HNK increased the expression of p62 and LC3 similar to CQ, and there is no obvious increase in the expression of p62 and LC3 when combining HNK and CQ.

Cathepsin D, a member of the lysosomal aspartyl cathepsin family, plays a crucial role in cell fate and tissue homeostasis through apoptosis (21). Based on the above results, HNK induced apoptosis, thus we investigated the effect of HNK on cathepsin D expression. In the A549 and H460 cells, HNK increased the level of cathepsin D protein in both a time-dependent and concentration-dependent manner. HNK at 20 µM increased expression of cathepsin D at 24 h after treatment in both cell lines (Fig. 8A). Upregulation of cathepsin D was observed even at 4 h post HNK treatment. The increase in cathepsin D by HNK was maintained for up to 24 h in the A549 and H460 cells (Fig. 8B).

In order to further determine the role of cathepsin D in the apoptosis-induction of HNK, cathepsin D siRNA and pepstatin A (PepA) as an aspartate protease inhibitor of cathepsin D were used in the experiments. As shown in Fig. 8C, the sirNA efficiently knocked down cathepsin D expression in the A549 and H460 cells with or without HNK treatment. Notably, the expression of PARP and cleaved caspase-3 was elevated after the gene of cathepsin D was knocked down in both cell lines treatment with HNK (Fig. 8C). In addition, PepA significantly potentiated the inhibitory effect on the proliferation of the A549 and H460 cells by HNK (Fig. 8D).