Introduction

Lung cancer was the most commonly diagnosed cancers among men and the second most commonly diagnosed cancers among women in 2015 in China [1]. According to the data from American Cancer Society, more than a quarter of all cancer deaths are due to lung cancer [2]. Most lung cancers are non-small cell lung cancer (NSCLC), so it is meaningful to find out drugs that can be candidates for the anti-cancer therapy of NSCLC.

Albendazole (ABZ) is a broad spectrum benzimidazole carbamate anthelminthic with low toxicity and is widely used in human and animals [3]. ABZ inhibits helminths cell proliferation by binding to β-tubulin and inhibiting microtubule polymerization [4]. As a result, it reduces parasite survival and reproduction. In 1985, it was reported that benzimidazole carbamates had anti-tumor effect in mouse leukaemia cells [5]. Subsequently, many researchers studied the anti-tumor activity of ABZ and found that it had anti-proliferative properties in many cancer cells, such as hepatocellular carcinoma cells [6], colorectal cancer cells [7]. Phase I clinical trial has been done to determine maximum tolerated dose of oral albendazole in patients with advanced cancer [8]. However, the inhibitory mechanism of ABZ on NSCLC remains to be illustrated.

Tumor cells must change their metabolic way to meet their increasing needs for growth and proliferating. There is a famous phenomenon which is called Warburg effect in tumor glycometabolism. It describes the phenomenon that most cancer cells generate ATP by a high rate of glycolysis converting glucose to lactate, rather than metabolizing glucose by oxidative phosphorylation, even when oxygen is plentiful [9, 10]. Due to Warburg effect, tumor cells require much more glucose than normal cells and have highly active glycolysis. Lactate generated by glycolysis acidizes cells and benefits tumor.

Hypoxia-inducible factor-1α (HIF-1α) is the most important cell transcriptional activator in response to hypoxia and regulates a series of enzymes expression in the glycolytic pathway, such as succinate dehydrogenase [11], fumarate hydratase [11], pyruvate dehydrogenase kinases [11, 12], hexokinase (HK) [12], glucose transporters GLUT1 and GLUT3 [12, 13]. By anaerobic glycolysis, tumors metabolize glucose to lactate and survive in hypoxia environment. Under normoxic and iron-replete conditions, the protein expression of HIF-1α in cells is not detectable due to its rapid degradation [14]. However, the degradation is weak under hypoxia condition and results in the accumulation of HIF-1α in cells. Hyperactive metabolism in tumor cells increases oxygen consumption and leads to hypoxia. As a result, HIF-1α is accumulated in anoxic tumor cells. HIF-1α binds to vascular endothelial cell growth factor (VEGF) gene promoter and induces VEGF expression and angiogenesis, subsequently [15]. VEGF, usually overexpressed in NSCLC, is one of the most specific and crucial regulators of angiogenesis [16, 17], which suggests that targeting VEGF expression may an effective approach for NSCLC treatment. In this study, we explored the molecular mechanisms involving in the inhibition of glycolysis and VEGF expression affected by ABZ. These findings offered new insights into our understanding of the mechanisms underlying the anti-tumor function of ABZ.

Materials and methods

Cell culture and treatment

NSCLC A549 and H1299 cells were obtained from American Type Culture Collection (ATCC; Rockville, MD, USA), cultured in DMEM medium (Gibco, Grand Island, NY, USA), and supplemented with 10% FBS and 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco) at 37 °C in a 5% CO2 incubator. Cells were digested using 0.25% trypsin and passaged at 80–90% confluence. Before treatment, cells were allowed to attach the surface for 24 h. Cells were incubated with CoCl2 (300 µM) or DFX (0, 100, 200 µM) in the presence or absence of ABZ (500 nM) for 24 h, followed by the western blot analysis, qRT-PCR, enzyme activity measurement, and lactate detection. Cells were exposed to PX-478 (0, 20, and 50 µM) in the presence or absence of DFX (200 µM) for 24 h, and the levels of HIF-1α mRNA, HIF-1α protein, and VEGF protein, the enzyme activities of HK, PK, and lactate dehydrogenase (LDH), and production of lactate were determined.

HK, PK, and LDH activities and lactate content measurements

Cells were inoculated in 6-well plates. After drug treatment, cells were digested with 0.25% trypsin and washed with PBS. Cell suspension was homogenized on ice. Protein concentration in homogenate was quantified by the BCA Protein Assay Kit (Thermo Fisher Scientific). HK, PK, and LDH activities and lactate content were measured by colorimetric assay using specific test kit bought from Solarbio (Beijing, China) according to the manufacturer’s suggestions. The absorbance of HK and PK was measured at 340 nm and the absorbance of LDH was measured at 450 nm. The activities of HK, PK, and LDH were calculated according to the manufacturer’s suggestions. The activity of HK, PK, and LDH in control group was normalized to 1.0.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from treated cancer cells using Trizol reagent (Tiangen, China) according to the manufacturer’s instructions. Concentration and purity of total RNA samples were determined using the Nanodrop2000 (Thermo Scientific, Wilmington, DE, USA). Up to 5 µg of RNA was reverse transcribed into cDNA using the miScript II Reverse Transcription Kit (Qiagen, Germany) according to the manufacturer’s protocols. The following primers were used for RT-PCR: HIF-1α (forward: 5′-TGC TCA TCA GTT GCC ACT TC-3′, reverse: 5′-CCA AGC AGG TCA TAG GTG GT-3′), and β-actin (forward: 5′-CTC CAT CCT GGC CTC GCT GT-3′, reverse: 5′-GCT GTC ACC TTC ACC GTT CC-3′). RT-PCR was performed using a miScript SYBR Green PCR Kit (Qiagen). β-actin mRNA was used as the reference gene to normalize the gene expression. Relative levels of mRNA were calculated using 2− ΔΔCt method. All reactions were performed in triplicate.

Western blot analysis

For protein analysis, harvested cell pellets were lysed in RIPA lysis buffer (Beyotime, Haimen, China) supplemented with 1 mM PMSF. Cell lysates were centrifuged at 12,000×g at 4 °C for 5 min, and concentration of proteins in supernatant was quantified by the BCA Protein Assay Kit (Thermo Fisher Scientific). Samples with equal proteins were electrophoresed with SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with 5% skim milk and incubated with monoclonal anti-HIF-1α antibody (Abcam, Cambridge, UK) or anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h at room temperature, and then with HRP-conjugated goat anti-rabbit IgG (Abcam) for 1 h. β-actin was used as an internal control. Proteins were visualized by ECL (Beyotime).

Xenograft tumor model

Four-to-five-week-old male athymic nude BALB/c mice (SLAC Laboratory, Shanghai, China) were used in this study. All mice were housed in a pathogen-free environment with ad arbitrium supply of water and food according to the guideline of Animal Care and Ethics Committee of Henan University (Kaifeng, China), and all experiments were performed in accordance with the protocols approved by the committee of Huaihe Hospital of Henan University. A549 xenografts were developed by subcutaneous injection of the A549 cells (2 × 106 cells per animal) into the flank of nude mice. The mice were randomly divided into two groups of 6 animals. Group 1 (the control group) received vehicle and Group 2 (the ABZ group) was administered 50 mg/kg of ABZ. Both groups received the treatment intraperitoneally every 3 days. Tumor growth was monitored by caliper, and tumor volumes were calculated every 5 days using the formula: Tumor volume = length × width2/2. After 20 days, the animals were sacrificed and the tumors were harvested and weighted. Tumor tissues were stored at −80 °C for subsequent RNA and protein extraction and enzyme activity measurements.

Data statistics

All data were analyzed using student’s t test or one-way analysis of variance (ANOVA). Data were considered to be statistically significant if P < 0.05.

Results

ABZ inhibits hypoxia-induced HIF-1α expression and VEGF expression in NSCLC cells

VEGF is a specific and crucial regulator of angiogenesis, which is promoted by HIF-1α and is a key process involved in tumor progression [18–20]. In order to examine whether ABZ inhibits expression of HIF-1α and VEGF in A549 cells, we treated cells with CoCl2, a hypoxia-mimetic agent, in the presence or absence of ABZ for 24 h. HIF-1α mRNA and protein expression levels of HIF-1α and VEGF were detected by western blot. The results showed that ABZ did not change the expression of HIF-1α mRNA and protein levels of HIF-1α and VEGF in A549 cells. CoCl2 significantly increased the expression of HIF-1α mRNA and protein levels of HIF-1α and VEGF, which could be reduced by ABZ in A549 cells (Fig. 1a–d). To confirm the above effects of ABZ, A549 cells were treated with different concentration of DFX, another hypoxia-mimetic agent, in the presence or absence of ABZ for 24 h. As shown in Fig. 1e–h, DFX elevated the expression of HIF-1α mRNA and protein levels of HIF-1α and VEGF in A549 cells. Nevertheless, ABZ inhibited DFX-induced HIF-1α and VEGF expression in A549 cells. Similar findings were obtained in another NSCLC cell line H1299 (Fig. 1i–l). Collectively, our data revealed that ABZ had an ability to inhibit hypoxia-induced HIF-1α and VEGF expression.

Fig. 1
figure 1

ABZ inhibited hypoxia-induced HIF-1α and VEGF expression in A549 cells. a–d ABZ inhibited CoCl2-induced HIF-1α mRNA expression and the protein levels of HIF-1α and VEGF. A549 cells were treated with 300 µM of CoCl2 in the presence or absence of 500 nM of ABZ for 24 h. HIF-1α mRNA was detected by qRT-PCR, and protein expression levels of HIF-1α and VEGF were detected using western blot. ABZ inhibited DFX-induced HIF-1α expression and VEGF expression A549 (e–h) and H1299 (i–l) cells. Cells were treated with different concentrations of DFX (0, 100, and 200 µM) for 24 h. To assess the effect of ABZ on DFX-induced HIF-1α expression and VEGF expression, cells were co-incubated with 200 µM of DFX and 500 nM of ABZ for 24 h, followed by qRT-PCR and western blot analysis. Data are shown as mean ± standard deviation (SD; n = 4). NS not significant. *P < 0.05, **P < 0.01, ***P < 0.001

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ABZ inhibits hypoxia-induced glycolysis in NSCLC cells

Glycolysis is a major energy source in tumor and HIF-1α affects glycolysis by regulating a serious enzyme related to the glycolytic pathway [11]. To clarify whether ABZ had an effect on cellular glycolysis, A549 cells were treated with CoCl2 in the presence or absence of ABZ for 24 h. Relative activities of HK, PK, LDH, and the lactate production were measured by colorimetric analysis. As shown in Fig. 2a–d, ABZ inhibited CoCl2-induced activities of HK, PK, LDH, and lactate production in A549 cells. To confirm the above effects of ABZ, we also studied the effects of ABZ on DFX-induced glycolysis in A549 and H1299 cells. As expected, ABZ inhibited DFX-induced activities of HK, PK, LDH, and production of lactate in A549 (Fig. 2e–h) and H1299 (Fig. 2i–l) cells. Since HK, PK, and LDH are important enzymes in glycolysis and lactate is the major product of glycolysis, the result indicated that ABZ inhibited the glycolysis pathway in NSCLC cells.

Fig. 2
figure 2

ABZ inhibited hypoxia-induced glycolysis in A549 cells. a–d ABZ inhibited CoCl2-induced activities of HK, PK, and LDH and lactate production in A549 cells. A549 cells were treated with 300 µM of CoCl2 in the presence or absence of 500 nM of ABZ for 24 h. The activities of HK, PK, and LDH and lactate production were detected by colorimetric assay using specific test kit. ABZ inhibited DFX-induced activities of HK, PK, and LDH and lactate production in A549 (e–h) and H1299 (i–l) cells. Cells were treated with 200 µM of DFX in the presence or absence of 500 nM of ABZ for 24 h. Data are shown as mean ± standard error (SEM; n = 3). HK hexokinase, PK pyruvate kinase, LDH lactate dehydrogenase. NS not significant. *P < 0.05, **P < 0.01, ***P < 0.001

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Blocking HIF-1α inhibits glycolysis and VEGF expression

To confirm whether glycolysis and VEGF expression were regulated by HIF-1α, A549 cells were treated with 20 and 50 µM of PX-478, a HIF-1α inhibitor, for 24 h under hypoxia. We found that expression levels of HIF-1α mRNA and protein levels of HIF-1α and VEGF were declined with the increase of PX-478 concentration (Fig. 3a–d), indicating that blocking HIF-1α inhibited VEGF expression in NSCLC A549 cells. The relative activities of HK, PK, LDH, and the production of lactate were also decreased in the presence of PX-478 (Fig. 3e–h), suggesting that blocking HIF-1α inhibited glycolysis in NSCLC A549 cells.

Fig. 3
figure 3

Blocking HIF-1α inhibited glycolysis and VEGF expression. a–d PX-478, a HIF-1α inhibitor, significantly inhibited HIF-1α and VEGF expression. A549 cells were treated with 200 µM of DFX and different concentrations of PX-478 for 24 h. HIF-1α mRNA was detected using qRT-PCR and protein expression levels of HIF-1α and VEGF were detected using western blot. Data are shown as mean ± SD (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. e–h PX-478 inhibited DFX-induced activities of HK, PK, and LDH and lactate production in A549 cells. A549 cells were treated with 200 µM of DFX and different concentrations of PX-478 for 24 h. Data are shown as mean ± SEM (n = 3). HK hexokinase, PK pyruvate kinase, LDH lactate dehydrogenase. *P < 0.05, **P < 0.01, ***P < 0.001

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ABZ inhibits A549 xenograft growth in vivo

We further examined the effect of ABZ on HIF-1α and VEGF expression and glycolysis in A549 xenografts in nude mice. The mice were randomly divided into two groups of 6 animals. The control group received vehicle and the ABZ group was administered 50 mg/kg of ABZ. As illustrated in Fig. 4a, b, ABZ significantly inhibited tumor volume and weight in nude mice. ABZ treatment suppressed the expression of HIF-1α mRNA compared with the control group (Fig. 4c). The expression levels of HIF-1α and VEGF in A549 xenografts in the ABZ group were decreased compared with those in the control group (Fig. 4d). Furthermore, ABZ reduced the enzyme activities of HK, PK, and LDH compared with the control group (Fig. 4e). Taken together, these findings displayed that ABZ inhibited glycolysis and VEGF expression in vivo.

Fig. 4
figure 4

ABZ inhibited A549 xenograft growth in mouse models. a, b ABZ inhibited the tumor volume and weight of A549 xenograft growth in mouse models. A549 were subcutaneously injected into the flank of nude mice. The control group received vehicle and the ABZ group was administered 50 mg/kg of ABZ. Both the groups received the treatment intraperitoneally every 3 days, and tumor volume was measured and calculated every 5 days. After 20 days, the animals were sacrificed and the tumors were harvested and weighted. c, d ABZ inhibited HIF-1α mRNA and protein and VEGF expression in A549 xenografts in mouse models. HIF-1α mRNA was detected using qRT-PCR and protein expression levels of HIF-1α and VEGF were detected using western blot. e ABZ decreased the activities of HK, PK, and LDH in A549 xenografts. The activities of HK, PK, and LDH were detected by colorimetric assay using specific test kit. Data are shown as mean ± SD (n = 6). HK hexokinase, PK pyruvate kinase, LDH lactate dehydrogenase. *P < 0.05, **P < 0.01, ***P < 0.001

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Discussion

ABZ has a wide effect on tumor development, such as inhibiting tumor growth, suppressing proliferation, arresting cell cycle, and promoting cells apoptosis [21–23]. Angiogenesis, an essential process for tumor growth, offers nutrient for tumor, takes metabolic waste away, and provides support for tumor spread and metastasis, and tumor frequently overexpresses pro-angiogenic factors, such as VEGF, for their progression [24–26]. Our result showed that ABZ inhibited hypoxia-induced accumulation of HIF-1α and VEGF expression. Noorani et al. [27] reported that nanoscale ABZ enhanced the anti-angiogenic property by inhibition of VEGF in ovarian cancer in both in vitro and in vivo models. Pourgholami et al. [28] found that tumoral HIF-1α and VEGF protein levels were highly suppressed in ABZ-treated mice. ABZ abolished tumor angiogenesis and ascites formation by inhibiting VEGF production in nude mice-bearing OVCAR-3 peritoneal tumors [29]. We speculated that the reduced VEGF expression is the result of ABZ-induced HIF-1α inhibition.

HIF1α has been suggested to be overexpressed in NSCLC and targeting the HIF pathway may prove of importance in the treatment of NSCLC [30]. CoCl2 and DFX can block the HIF-1α degradation thus inducing the accumulation of HIF-1α and angiogenesis in tumor cells [31]. Borcar et al. found that CoCl2 and DFX increased HIF-1α expression in a dose-dependent manner [32], which is consistent with our study. PX-478 suppresses the levels of hypoxia-induced HIF-1α in cancer cells [33, 34]. Our result showed that PX-478 inhibited HIF-1α and resulted in reduction of VEGF expression in NSCLC A549 cells, which demonstrated that VEGF expression is HIF-1α-dependent in NSCLC A549 cells. Targeting HIF-1α may be a potential treatment for NSCLC.

Anaerobic glycolysis is important for the energy production and low-pH environment maintenance in tumor. Luo et al. [35] found that HIF-1α acts as a switch for oxygen regulation in glucose metabolism by regulating the expression and activity of pyruvate kinase muscle isozyme 2 (PKM2) in cancer cells. There are some critical enzymes in glycolysis, such as HK, LDH, and PK. HK catalyzes six-carbon sugars into hexose phosphate and is a rate-limiting enzyme in glycolysis. LDH converts pyruvate to lactate and acidizes the tumor microenvironment, which would benefit the tumor. PK catalyzes the transfer of phosphoenolpyruvate into pyruvate, which is upstream of the decisive point for glycolytic or oxidative metabolism [36]. Inhibition of glycolysis can block the energy source of tumor cells thus suppressing the tumor development. Sanchez et al. [37] found that dichloroacetate induced growth reduction in multiple myeloma cells by inhibiting aerobic glycolysis. In lung cancer cells, p53 suppressed hypoxia-stimulated glycolysis through Ras-related associated with diabetes (RRAD), which inhibits GLUT1 translocation to the plasma membrane and represses glycolysis [38]. Knockdown glycometabolic proteins using siRNA could also downregulate glycolysis and inhibit proliferation of tumor cells [39]. It is reported that ABZ not only blocks the ATP formation by inhibiting the fumaric reductase system, but also depletes glycogen storage and inhibits glucose absorption in parasite [40, 41]. The exactly mechanism of ABZ inhibits glycolysis in tumor cells remains unknown. In our study, we found that ABZ suppressed HK, PK, LDH activities and lactate production in A549 cells, indicating glycolysis pathway in A549 cells was inhibited by ABZ. Furthermore, our results showed that inhibition of HIF-1α by PX-478 resulted in reduction of glycolysis. It demonstrated that HIF-1α could influence the activities of HK, PK, LDH, and the lactate production and glycolysis is HIF-1α-dependent in NSCLC A549 cells. We speculate that ABZ suppresses glycolysis by inhibiting HIF-1α expression.

In conclusion, our results suggested that ABZ inhibited HIF-1α-dependent glycolysis and VEGF expression in NSCLC cells. Our in vivo experiments supported that ABZ may be an important candidate for anti-cancer therapy of NSCLC. Researching on the inhibitory mechanism of ABZ on NSCLC lays a theoretical foundation for combination drug therapy.