Scutellarin induces apoptosis and autophagy in NSCLC cells through ERK1/2 and AKT Signaling Pathways in vitro and in vivo

2.1 Chemicals and Reagents

Scutellarin (purity ≥ 98%) was purchased from Sigma Aldrich (St. Louis, MO, USA), and dissolved in PBS (pH 7.4). Autophagy inhibitor hydroxychloroquine (HCQ), ERK1/2 inhibitor U0126 and AKT inihbitor MK-2206 were obtained from Selleck Chemicals LLC ( Houston, TX, USA ). The primary antibodies against LC3, p62, ERK1/2, p-ERK1/2, AKT, p-AKT, β-actin and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

2.2 Cell Lines and Cell Culture

Human NSCLC cell lines, including PC-9 and H1975, human cervical cancer cell line Hela, human hepatocellular carcinoma cell line HepG2, and human normal lung epithelial cell line Beas-2B were provided by Shanghai Life Sciences Research Institute Cell Resources Center. All cell lines were maintained in RPMI (Roswell Park Memorial Institute medium) 1640 containing 10% FBS (fetal bovine serum), and incubated at 37 ℃ in a humidified atmosphere with 5% CO₂.

2.3 Cell Viability Assay

Cell viability was examined by the MTT assay. In brief, PC-9, H1975, HepG2, Hela and Beas-2B cells were seeded in 96-well plates (4×10³cells/well), and cultured overnight. After 24 or 48 hours treatment of scutellarin (0, 5, 10, 20, 40, 80, 160 μM), cells were subjected to 20 μL of MTT solution (5 mg/mL), followed by addition with 150 μL of dimethyl sulfoxide (DMSO). The optical density (OD) at wavelength of 490 nm was detected by a Multiskan Ascent Revelation Plate Reader (Thermo, USA).

2.4 Detection of cell apoptosis

Cell apoptosis was measured by flow cytometry using the Annexin V-FITC/PI Apoptosis Kit (MultiSciences Biotech Co., Ltd). Briefly, PC-9 and H1975 cells were exposed to 160 μM scutellarin for 48 hours. After treatment, cells were harvested and washed with ice-cold PBS, then resuspended in 500 μL binding buffer. Subsequently, cells were stained using 5 μL Annexin V-FITC and 10 μL PI solution, followed by incubation for 5 mins at 37 ℃ in a dark atmosphere. Finally, the number of apoptotic cells was examined by FACS (Becton-Dickinson, Bedford, MA, USA).

2.5 Western blot analysis

Cells were lysed in a standard RIPA buffer supplemented with 1% protease and phosphatase inhibitors. Protein concentration was measured by the BCA assay. Equal amounts of proteins were separated by 8%-12% SDS-PAGE, then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skimmed milk for one hour, the membranes were overnight incubated with primary antibodies, followed by incubation with anti-rabbit secondary antibody for another one hour. Immunoreactivity was visualized utilizing an enhanced chemiluminescence (ECL) system, and protein bands were quantified by the Image-Pro Plus 6.0.

2.6 In vivo xenograft and treatment experiments

Female BALB/c nude mice were purchased from Guangdong Medical Laboratory Animal Center (Fushan, Guangdong, China). The animal procedures were approved by the Animal Care and Use Committee of Guangdong Provincial Hospital of Chinese Medicine (the Ethics Approval Number 2016023) and the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals. To establish xenograft tumor model, mice were subcutaneously injected with 4×10⁶ H1975-Luciferase cells (stably expressing fluorescence) suspended in 200 μL PBS. When tumor reached approximately 100 mm³ (Volume = Length ×width² × 0.5), mice were randomly divided into three groups (n = 8): the vehicle; the low dose scutellarin (30 mg/kg); the high dose scutellarin (60 mg/kg). At this point, scutellarin was dissolved in PBS (PH 7.4), and was orally administered to each mouse once a day for three weeks. The tumor dimensions were measured using digital calliper every 3 days. Before treatment or after the last administration, the tumor size was monitored by in vivo bioluminescence imaging (IVIS Lumina LT Series III Pre-Clinical In Vivo Imaging System). After 3 weeks, all mice were humanely sacrificed and the tumors were resected for protein quantitation analysis.

2.7 Immunohistochemistry

Twenty surgically excised lung adenocarcinoma specimens and adjacent normal lung tissues were fixed in 4% paraformaldehyde at 4℃, then embedded in paraffin, and 4-μm paraffin sections were obtained. The sections were deparaffinized and serially rehydrated with xylene. The anti-gen retrieval was performed before the sections were incubated in 10% serum blocking solution. Then the slides were incubated with primary antibodies (p-AKT and p-ERK) in blocking solution overnight at 4 ℃. After washing and incubation with secondary antibody at room temperature for 30 m, sections were visualized with diaminobenzidine and couterstained with hematoxylin. Finally, these immune-stained slides were evaluated and scored by two independent pathologists.

2.8 Statistical Analysis

All data were presented as the mean ± SEM. The differences between the groups were analyzed using unpaired Student's t test or one-way ANOVA test in SPSS software (version 17.0). p < 0.05 was considered to be statistically significant.

3.1 Effects of scutellarin on the proliferation and apoptosis on NSCLC cell lines

To determine the anti-tumor effect of scutellarin on NSCLC cells, the MTT assay was firstly employed. PC-9 and H1975 cells were treated with various concentrations of scutellarin (0, 5, 10, 20, 40, 80, 160 μM) for 24 or 48 hours. As shown in Fig. 1B, treatment of scutellarin clearly inhibited cell growth in a dose and time-dependent manner. In addition, the anti-proliferation effects of scutellarin on cervical cancer Hela cells and hepatocellular carcinoma HepG2 cells were confirmed by MTT assay. We found that scutellarin inhibited the cell viability of HepG2 and Hela cells (Fig. 1C), however, NSCLC cells were more sensitive to scutellarin than hepatocellular carcinoma and cervical cancer cells. Of note, human normal lung epithelial cell line Beas-2B was involved to determine the toxicity of scutellarin by MTT assay, and results showed that scutellarin exhibited no significant cytotoxic activity on Beas-2B cells (Fig. 1D). Additionally, we detected the cell apoptosis by flow cytometry using the Annexin V-FITC/PI Apoptosis Kit. Results showed that 160 μM scutellarin treatment significantly induced apoptosis, when compared with the control cells (Fig. 1E). Thus, scutellarin displayed a marked anti-tumor response to NSCLC cells.

3.2 Scutellarin induced autophagy in NSCLC cells

Considering that autophagy plays an essential role in cancers, here, we therefore examined whether scutellarin was able to alter the expression of autophagy-related proteins. Microtubule-associated protein light chain 3 (LC3), an excellent marker of autophagy, is widely used for monitoring autophagy ²⁶. During autophagy induction, the transition of the non-lipidated form of LC3 (LC3-I) to the lipidated form of LC3 (LC3-II) is indispensable ²⁷. Thus, the increase of LC3-II level or LC3-II/LC3-I ratio specifically signifies the induction of autophagy. As expected, results showed that 160 μM scutellarin increased LC3-II conversion in PC-9 and H1975 cells (Fig. 2A). Thus, these results implied that scutellarin induced autophagy in NSCLC cells.

To further verify the role of autophagy in NSCLC cells, autophagy inhibitor HCQ was used. HCQ is generally utilized to inhibit autophagy by impairing the fusion of autophagosomes with lysosomes ²⁸. As shown in Fig. 2B, an increase in the level of LC3-II protein in PC-9 and H1975 cells treated with 5, 10, 20 μM HCQ was observed, while p62 expression was also up-regulated, suggesting that HCQ inhibited induction of autophagy. Consistently, 10 μM HCQ did not affect the cell viability of NSCLC cells (Fig. 2C). Thus, 10 μM HCQ was selected for further research. Here, we noted that 10 μM HCQ indeed enhanced the expression of LC3-II, and HCQ combined with scutellarin markedly increased the level of LC3-II, indicating that HCQ could block scutellarin-induced autophagy (Fig. 2D). We next examined whether HCQ treatment could affect the anti-proliferative function of scutellarin using MTT assay. As shown in Fig. 2E, 10 μM HCQ treatment had no significant effect on cell proliferation in PC-9 and H1975 cells. Interestingly, 10 μM HCQ slightly abolished the anti-proliferative ability of scutellarin, and the efficiency was better when the dose of scutellarin was higher. Collectively, our results indicated that scutellarin-induced autophagy is not cytoprotective, but antineoplastic.

It is noteworthy that autophagy and apoptosis might be connected with each other and occur simultaneously or sequentially under certain circumstances ²⁹. To investigate whether there exists interaction between autophagy and apoptosis, we examined the effect of autophagy inhibitor on scutellarin-induced apoptosis. Here, results showed that scutellarin plus HCQ treatment reduced scutellarin-induced apoptosis (Fig. 2F). Thus, scutellarin-induced autophagy promoted apoptosis, leading to cell death.

3.3 Activation of ERK1/2 signaling pathway is required for scutellarin-induced autophagy

Accumulating evidences suggest that ERK1/2 signalling pathway has recently emerged as an important player in regulating autophagy ¹⁵. To evaluate whether scutellarin-induced autophagy was associated with ERK1/2 signalling, we detected the level of p-ERK1/2 and ERK1/2 in PC-9 and H1975 cells treated with scutellarin. As shown in Fig. 3A, in accordance with LC3-II protein, scutellarin treatment dramatically increased the expression of p-ERK1/2, suggesting that elevated autophagy might be modulated by the ERK1/2 signalling. To exclude this possibility, we next determined the unclear relationship between autophagy and ERK1/2 using a specific ERK1/2 inhibitor, U0126. As exhibited in Fig. 3B, 20 μM U0126 attenuated p-ERK1/2 level, whereas total ERK1/2 showed no change, and scutellarin plus U0126 treatment decreased scutellarin-induced activation of ERK1/2. Moreover, scutellarin treatment caused autophagic responses in NSCLC cells, however, scutellarin in combination with U0126 remarkedly attenuated the expression of LC3-II. Overall, scutellarin triggered high autophagy partly by up-regulating ERK1/2 signaling pathway.

3.4 Scutellarin inhibited AKT signaling pathway, and there existed crosstalk between ERK and AKT

To further confirm whether scutellarin affects the AKT signaling, we treated H1975 cells with 0, 40, 80, 160 μM scutellarin. Results showed that scutellarin treatment increased AKT phosphorylation (p-AKT), whereas no difference was observed in the total AKT level (Fig. 4A). In addition, AKT inhibitor MK-2206 blocked p-AKT, and inhibition of AKT by MK-2206 induced autophagy (Fig. 4B), suggesting AKT pathway negatively regulated autophagy. Furthermore, we found that inhibition of ERK1/2 by U0126 increased the expression of p-AKT (Fig. 4C). Similarly, AKT inhibitor MK-2206 improved p-ERK1/2 level, indicating that there existed crosstalk between ERK1/2 and AKT pathways.

3.5 Scutellarin suppressed tumor growth in mouse xenograft model

To further investigate the anti-tumor activity of scutellarin in vivo, H1975-Luciferase cells were subcutaneously injected into BALB/c nude mice. As a result, mice treated with 30, 60 mg/kg scutellarin showed significant suppression of the tumor growth, compared with vehicle controls (Fig. 5A, 5D). Notably, 60 mg/kg scutellarin reduced about 50% of the tumor size, with better efficacy than 30 mg/kg scutellarin (Fig. 5B, 5C). In addition, we measured the expression of LC3, p-ERK1/2, ERK1/2, and p-AKT in tumors, and found that 30, 60 mg/kg scutellarin treatment could up-regulate LC3-II and p-ERK1/2 level, and down-regulate p-AKT (Fig. 5E). Taken together, these observations demonstrated that scutellarin suppressed tumor growth in mouse xenograft model, in accordance with in vitro cell experiments.

3.6 Clinical correlation of AKT and ERK in NSCLC

To clarify the clinical correlation of AKT and ERK in NSCLC, twenty surgically excised lung adenocarcinoma specimens and adjacent normal lung tissues were assessed using immunohistochemistry. As shown in Fig. 6, high-expressions of p-AKT and p-ERK were observed in lung adenocarcinoma specimens compared with normal lung tissues. Result of p-AKT expression was consistent with our in vitro cell experiment, indicating that AKT played as an oncogene. In most cases, activated ERK pathway is involved in directing cell proliferation, survival and apoptosis ¹². Here, we found that ERK was positively expressed in normal lung tissues, and significantly higher expressed in tumor tissues.