Baicalein suppresses non small cell lung cancer cell proliferation,invasion and Notch signaling pathway

Abstract

BACKGROUND:

Baicalein is an important Chinese herbal medicine and has multiple pharmacological activities. However, the biological mechanisms of the anti-tumor effects of Baicalein on non small cell lung cancer (NSCLC) still need to be understood.

METHODS:

Human NSCLC A549 and H1299 cells were pretreated with Baicalein or DMSO. Cells viability and transwell cell invasion assays were performed to assess cell proliferation and invasion. QRT-PCR assay was used to analyze mRNA expression levels of Twist1, E-cadhertin, Vimentin, Notch1 and hes-1. Western blot analysis was also performed to determine protein expression.

RESULTS:

In the study, we found that Baicalein had a significantly inhibited effect on proliferation ability of A549 and H1299 cells. Cells treated with Baicalein showed a down-regulated expression of CyclinD1 and CDK1 in A549 and H1299 cells. Furthermore, we found that Baicalein significantly inhibited cell invasion and Epithelial-Mesenchymal Transition (EMT) by up-regulating the mRNA and protein expression of E-cadherin and down-regulated the Twist1 and Vimentin expression, Moreover, Treatment of Baicalein down-regulated Notch1 and hes-1 expression in A549 and H1299 cells, which indicated that Baicalein could suppress the Notch signaling pathway.

CONCLUSION:

Our studies suggest that Baicalein may be a potential phytochemical flavonoid for therapeutics of NSCLC and serve as a molecular target for NSCLC.

1. Introduction

Lung cancer is one of the most common malignant cancers and non small cell lung cancer accounts for about 80% of all lung cancer cases [1]. Although some advances in diagnosis and therapeutic methods of NSCLC, the 5-year survival rate of NSCLC patients remain lower due to tumor metastasis and recurrence [2, 3]. Thus, exploring novel diagnostic biomarkers and novel targets for NSCLC treatment are necessary.

Baicalein is an important Chinese herbal medicine and has multiple pharmacological activities including antibacterial, antiviral, anti-inflammatory, antioxidant and antitumor activities [4, 5, 6]. For example, studies show that Baicalein inhibits cells epithelial to mesenchymal transition by down-regulation of Cyr61 and LOXL-2 expression in breast cancer cells [7]. Baicalein inhibits the migration and invasion of B16F10 mouse melanoma cells through inactivation of the PI3K/Akt signaling pathway [8]. In NSCLC, Zheng et al demonstrates that Baicalein inhibits the migration and invasion of B16F10 mouse melanoma cells through inactivation of the PI3K/Akt Signaling Pathway [9]. Cathcart et al also found that Baicalein has a large anti-cancer effect of in non-small cell lung cancer in-vitro and in-vivo [10]. However, the biological mechanisms of the anti-tumor effects for Baicalein in non small cell lung cancer (NSCLC) still need to be verified.

In the study, we found that Baicalein had significantly inhibited effects on cell proliferation, invasion and Epithelial-Mesenchymal Transition (EMT) in NSCLC. Treatment of Baicalein in NSCLC cells suppressed Notch signaling pathway by decreasing Notch1 and hes-1 expression. Thus, our studies suggest that Baicalein may be a potential phytochemical flavonoid for therapeutics of NSCLC and serve as a molecular target for NSCLC.

2. Materials and methods

2.1 Cell lines culture

Human NSCLC cell lines A549 and H1299 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and added with streptomycin (100 $\mu$ g/ml) and penicillin (100 U/ml). Baicalein was obtained from Cayman Chemical (Ann Arbor, MI, USA) and the DMSO was used as the control group.

2.2 Cell viability assay

The cell viability was determined by Cell Counting Kit-8 assay kit (CCK-8, Dojindo, Kumamoto, Japan). Briefly, NSCLC cells at a density of 3000 cell/well were seeded into 96-well plates. Concentrations of Baicalein (0, 20, 40, 60, 80, 100 $\mu$ M/L) were added in the plate and incubated for 24, 48, and 72 h. The cells were subsequently treated with 20 $\mu$ l of Cell Counting kit-8 reagents and incubated at 37 ${}^{\circ }$ C for an additional 2 h according to the manufacturer’s instructions. The absorbance values were determined at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

2.3 Cell invasion assay

NSCLC cells treated with Baicalein (80 $\mu$ M/L) or DMSO group NSCLC for 24 h, cells added with 200 $\mu$ l medium without FBS were pretreated seeded in the upper chamber. The 500 $\mu$ l medium supplemented with 10% FBS were added in the lower chamber and acted as a chemotactic agent. After cells were incubated at 24 h, the cells on the lower chamber were fixed using 4% paraformaldehyde for 10 min and stained with crystal violet. Cells were calculated in five random filed under a microscope.

Figure 1.

Baicalein inhibits cell viability of A549 and H1299 cells. (A)–(B) Cell viability was measured by CCK8 assay. The Concentrations of Baicalein (0, 20, 40, 60, 80, 100 $\mu$ M/L) and cells were incubated for 24, 48, and 72 h in A549 and H1299 cells. (C)–(D) The relative protein expression of CyclinD1 and CDK1 after cells were pretreated with Baicalein (80 $\mu$ M/L) compared with control group in A549 and H1299 cells. Values represent the mean $\pm$ SD of three independent experiments. ${}^{*}P<$ 0.05.

2.4 RNA extraction and quantitative real time polymerase chain reaction (qRT-PCR)

Total cell RNA was extracted using the using TRizol reagent (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. The RNA was reversed to first-strand cDNA by the Reverse Transcriptase Kit (TAKARA, Tokyo, Japan). SYBR Green Mixture was used for the qRT-PCR reactions (TAKARA, Tokyo, Japan) on the ABI 7500 System. Primers sequences were synthesized by TAKARA (Dalian, China) as follow: Twist1-forward: 5’-AGAAGTCTGCGGGCTGT GGCG-3’, Twist1-reverse: 5’-GAGGGCAGCGTGGG GATGATC-3’; $\beta$ -actin-forward: 5’-AGTGTGACGTG GACATCCGCAAAG-3, $\beta$ -actin-reverse: 5’-ATCCAC ATCTGCTGGAAGGTGGAC-3’. E-cadherin-forward: 5’-CGAGAGCTACACGTTCACGG-3’, E-cadherin-reverse: 5’GGGTGTCGAGGGAAAAATAGG-3’. Vimentin-forward: 5’-GACGCCATCAACACCGAGTT-3’, Vimentin-reverse: 5’-CTTTGTCGTTGGTTAG CTGGT-3’. The mRNA expression quantification was analyzed using the 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }Ct}$ method and mRNA expression was normalized to GAPDH.

Figure 2.

Baicalein inhibits cell invasion ability and epithelial-mesenchymal transition of A549 and H1299 cells. (A)–(B) The cell invasion assay and invasive cell number was showed after cells were pretreated with Baicalein (80 $\mu$ M/L) compared with control groups in A549 and H1299 cells. (C)–(D) The relative mRNA expression of Twist1, E-cadherin, and Vimentin was showed after cells were pretreated with Baicalein (80 $\mu$ M/L) compared with control groups in A549 and H1299 cells. (E)–(F) The relative protein expression of Twist1, E-cadherin, and Vimentin was showed after cells were pretreated with Baicalein (80 $\mu$ M/L) compared with control groups in A549 and H1299 cells. Values represent the mean $\pm$ SD of three independent experiments. ${}^{*}P<$ 0.05.

Figure 3.

Baicalein inhibits Notch signaling pathway by regulating Notch1 and hes-1 expression. (A)–(B) The relative mRNA expression of Notch1 and hes-1 was showed after cells were pretreated with Baicalein (80 $\mu$ M/L) compared with control groups in A549 and H1299 cells. (C)–(D) The relative protein expression of Notch1 and hes-1 was showed after cells were pretreated with Baicalein (80 $\mu$ M/L) compared with control groups in A549 and H1299 cells. Values represent the mean $\pm$ SD of three independent experiments. ${}^{*}P<$ 0.05.

2.5 Western blot analysis

The NSCLC cells were lysed and extracted using lysis buffer (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). The protein concentration in the supernatants was determined using a BCA kit (Beijing Biosynthesis Biotechnology Co., Ltd.). Equal amount of protein was separated using 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were incubated with antibodies against Twist1 (1:1000, CST, USA), N-cadherin (1:1000, CST, USA) and Vimentin (1:1000, CST, USA), Notch1 (1:1000, CST, USA), hes-1 (1:1000, CST, USA) and GAPDH (1:2000, Santa Cruz Biotechnology, USA) for 4 ${}^{\circ }$ C overnight. Subsequently, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (CST, USA) at 37 ${}^{\circ }$ C for 1 h. The membranes were washed again and transferred to freshly made ECL solution (Immobilon Western; Millipore, Billerica, MA, USA) and observed, recorded the signals using the Gel Imagine System (Bio-Rad, Hercules, CA, USA).

2.6 Statistical analysis

Data are presented as the mean $\pm$ standard deviation. The data were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). $P<$ 0.05 was considered to indicate a statistically significant difference.

3. Results

3.1 Baicalein inhibits cell proliferation ability of A549 and H1299 cells

The cells were treated with Baicalein (0, 20, 40, 60, 80, 100 $\mu$ M/L), CCK8 cell assays were performed to assess the cell viability in A549 and H1299 at 24 h, 48 h, and 72 h. The results demonstrated that cell viability was significantly inhibited by Baicalein in a dose-and time-dependent manner (Fig. 1A and B). The higher inhibited efficiency was 80 $\mu$ mol/L after cells were treated at 48 h. Furthermore, western blot assays showed that cell proliferation associated protein CylinD1 and CDK1 was inhibited by treating with Baicalein (80 $\mu$ mol/L) at 48 h, compared with control groups in A549 and H1299 cells (Fig. 1C and D). Thus, Baicalein could inhibit cell proliferation ability of A549 and H1299 cells.

3.2 Baicalein inhibits cell invasion ability and epithelial-mesenchymal transition of A549 and H1299 cells

We further examined the effect of Baicalein on cell invasion in A549 and H1299 cells. After cells were treated with Baicalein (80 $\mu$ mol/L) at 48 h, the cell invasion assay showed that cell invasion number was inhibited compared with the control group in A549 cells (Fig. 2A). Similar results were found in H1299 cells (Fig. 2B). The mRNA and protein expression levels of the marker proteins associated with the process of epithelial-mesenchymal transition (EMT) were detected using quantitative RT-PCR and Western blot assay. After cells were treated with Baicalein (80 $\mu$ mol/L) at 48 h, we found that Baicalein inhibited the cell Epithelial-Mesenchymal Transition (EMT) process by upregulating the E-cadherin and downregulated the Twist1, N-cadherin and Vimentin expression levels compared with control groups in A549 cells (Fig. 2C and E). Consistent with above results, Baicalein inhibited H1299 cells EMT process (Fig. 2D and F). Thus, these results demonstrated that Baicalein inhibited cell invasion and EMT process in NSCLC cells.

3.3 Baicalein inhibits Notch signaling pathway by regulating Notch1 and hes-1 expression

Several researchers have found that Notch signaling pathway associated with tumor EMT phenotype, which triggers cell invasion [11]. Activating Notch signaling pathway could also induce the EMT program by regulating transcription factors such as Twist1, ZEB1/2, and Snail1/2 in some tumor types [12]. Thus, we explored whether aberrant Baicalein could affect Notch1 and hes-1 expression levels in NSCLC cells. The results demonstrated that the mRNA and protein expression levels of Notch1 and hes-1 were downregulated after cells were treated with Baicalein (80 $\mu$ mol/L) at 48 h (Fig. 3A and D). Thus, the above results indicated that Baicalein inhibited Notch signaling pathway by regulating Notch1 and hes-1 expression.

4. Discussion

Baicalein showed the anti-tumor effects in some cancer types. Liu et al. reported that Baicalein suppresses the proliferation of acute T-lymphoblastic leuk-emia Jurkat cells by inhibiting the Wnt $\beta$ -catenin signaling [13]. Gade et al revealed that Baicalein could inhibit breast cancer MCF-7 cell proliferation in vitro, induces radiosensitivity, and inhibits hypoxia inducible factor [14]. Baicalein inhibits hepatocellular carcinoma cells through suppressing the expression of CD24 [15]. In the previous study, Baicalein was found to act as tumor suppressor in NSCLC cells, such as, Baicalein induces caspase-dependent apoptosis associated with the generation of ROS and the activation of AMPK in human lung carcinoma A549 cells [16]. Baicalein induced cell cycle arrest and apoptosis in human lung squamous carcinoma CH27 cells [17]. Baicalein increases cisplatin sensitivity of A549 lung adenocarcinoma cells via PI3K/Akt/NF- $\kappa$ B pathway [18]. In the study, we found that Baicalein significantly inhibited cell proliferation ability of A549 and H1299 cells. The protein expression levels related to cell proliferation CyclinD1 and CDK1 were downregulated after cells were treated with Baicalein in A549 and H1299 cells. Additionally, we demonstrated that Baicalein suppressed cell invasion ability and epithelial-mesenchymal transition of A549 and H1299 cells.

Aberrant transduction of Notch signaling was involved in many diseases including cancers [19]. Notch signaling pathway activators could promote cell invasion and induce the EMT program by regulating transcription factors and EMT makers [20]. In the previous study, Wang et al showed that the autonomous notch signal pathway is activated by Baicalein in K562 cells [21]. Treatment with Baicalein at 48 h, we demonstrated that the mRNA and protein expression levels of Notch1 and hes-1 were downregulated after treated with Baicalein at 48 h. Thus, the above results indicated that Baicalein inhibits Notch signaling pathway by regulating Notch1 and hes-1 expression.

In conclusion, we demonstrated that Baicalein could inhibit cell proliferation and invasion of NSCLC. Moreover, Baicalein suppressed cell EMT phenotype and inhibited Notch signaling pathway, which could inhibit tumor cells invasion. Thus, our study suggested that Baicalein may be a potential phytochemical flavonoid for therapeutics of NSCLC and serve as a molecular target for NSCLC.