The human lung cancer H1650 and H1299 cell lines were purchased from ATCC, which were cultured with RPMI-1640 medium containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin, which were maintained in the incubator with 5% CO₂ at 37°C. 1 × 10⁶ cells/ml were planted in 6-cm culture plates. Also, the plasmids were transfected into cells using Lipofectamine 2000 (#2173184) (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions.

The main reagents and antibodies used in this article are as follows: baicalin (>98%) (#B20570) was purchased from Shanghai Yuanye Biological Co., Ltd. Antibodies against AKT (#4691T), p-AKT (#2853T), CDK2 (#2546), CDK4 (#12790), Cyclin E2 (#4132), Bax (#5023T), Bcl-2 (#4223), and α-tubulin (#2125) were purchased from Cell Signaling Technology.

The cell viability of H1299 and H1650 with the baicalin treatment was determined using CCK8 (#MA0218) (meilunbio). The 96-well plates were planted were 5 × 10⁴/ml cells per well, which was treated with baicalin (0, 50, and 100 μg/ml) for different times. After treatment, the cell viability was measured at 450 nm wavelength.

A density of 3 × 10³/ml cells was planted in 10-cm plates. The cells after being treated with baicalin were then maintained for about 2 weeks until they grew to visible colonies. Then, the colonies were fixed with 4% paraformaldehyde and stained with crystal violet.

The 6-cm dish was seeded with 5 × 10⁵ cells then given different treatments for 24 h. Then subsequently, the cells were stained using a cell cycle staining kit (#CCS012) purchased from MultiSciences (Lianke) Biotech Co., Ltd., for 30 min in a dark room.

The percent of cell apoptosis was assayed using the FITC Annexin V Apoptosis Detection kit according to the instructions (#556547) (BD, United States).

The cells from the control and treatment groups were collected and lysed by RIPA. After being centrifuged at a high speed, the supernatant was collected. The protein concentrations were measured, then the total protein was separated by SDS-PAGE (8–12%) and transferred to a PVDF membrane. The PVDF membrane was blocked, and the primary antibody was incubated. Finally, the secondary antibody was incubated and detected by ECL Prime Western Blotting Reagent (#RPN2232) (Amersham).

Female BALB/c-nu mice were purchased and raised for a week to adapt to the environment, then the subcutaneous tumor model was established by subcutaneous injection of tumor cells with the final concentration at 5 × 10⁶/ml and subcutaneous injection in the underarm. After the tumor was formed, mice were randomly divided into four groups: control group (100 μl, 5% DMSO), baicalin group (100 μl, 20 mg/kg baicalin diluted with 5% DMSO), SC79 group, and SC79 combined use of baicalin group. Mice were treated for 16 days, and then the tumors were collected and fixed. The experiment was approved by the Animal Care and Use Committee of Zhejiang University of Traditional Chinese Medicine (approval ID: 11139).

Tumor tissues were fixed with for 24 h with 10% neutral-buffered formalin. Then, tissues were sectioned (4 μm) and sodium citrate buffer was used for the antigen retrieval of sections. Then, the sections were incubated with 10% normal goat serum and primary antibody for 1 h and overnight, respectively. Next, the biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, United States; catalog # BA-1000; dilution 1:200) was used to incubate the sections for 1 h and subsequently incubate with avidin–biotin-horseradish peroxidase (VECTASTAIN^® Elite ABC kit; Vector Laboratories; Burlingame, CA, United States; catalog # PK-4010) for 1 h. Finally, color was detected using DAB substrate kit (Vector Laboratories, catalog # SK-4100).

All tests were performed for three times in all studies unless otherwise stated. All data were expressed as mean ± SD. Student’s t-test was employed for the significance calculate between different groups.

To test the biological function of baicalin in lung cancer, we first analyzed the cytotoxicity and growth inhibition effects of baicalin on lung cancer cells H1299 and H1650. After the treatment with various concentrations of baicalin at three time points, the results indicated that cell viability of lung cancer cells was inhibited by baicalin and the efficacy was positively correlated with drug dose and treatment time (Figure 1A). Meanwhile, light microscopy observation also showed that baicalin significantly inhibited the viability of these two lung cancer cells compared with their controls (Figure 1B), with more detached and shrunken cells appearing.

To obtain the effect of baicalin on apoptosis, the cell viability was detected by flow cytometry. As shown in Figure 2A, the proportion of apoptotic cells significantly increased in the baicalin treatment group, compared with the control. Colony formation assay was used to determine the anti-proliferation effect of baicalin. Moreover, the colony forming ability was remarkably inhibited by baicalin treatment, even with low concentrations such as 5 μg/ml (Figure 2B), indicating that baicalin treatment inhibited cell proliferation of lung cancer. The mechanism in which baicalin inhibited the lung cancer cell proliferation was further investigated via flow cytometry to detect whether baicalin could cause cell cycle arrest. Our data showed that cell cycle was remarkably arrested in the G1/S phase, accompanied with a decreased G2/M phase (Figure 2C). Taken together, we found that the cell-cycle arrest and apoptosis induction might be the major antitumor mechanism of baicalin in lung cancer cells.

Furthermore, the results from western blotting in Figure 3A show that the protein expression level of Bax was significantly upregulated in a dose-dependent manner, whereas Bcl-2 expression levels were remarkably decreased. The mitochondrial apoptosis proteins Bax and Bcl-2 can be activated by caspase-9 (Venteo et al., 2010); therefore, caspase-9 expression was also detected by western blotting in both baicalin treatment group and control group. Also, the results showed that activated caspase-9 was remarkably upregulated in a dose-dependent manner after baicalin treatment, when compared with their controls (Figure 3A).

As we know, cell cycle progression is controlled by a cyclin component and several cyclin-dependent kinases (CDKs), including CDK4/6-cyclin D and CDK2-cyclin E (Phan and Croucher, 2020). The above flow cytometric analysis shows that baicalin treatment could induce G1/S phase arrest. Thus, western blotting was performed to detect whether these cell cycle mediators were regulated by baicalin treatment, and we found that the proteins including CDK2, CDK4, and Cyclin E2 were decreased significantly after baicalin treatment (Figure 3B).

Akt/mTOR is a classical pathway involved in numerous cellular functions, including cellular proliferation, cell cycle progression, and the development of cancer (American Association for Cancer Research, 2017; Janku et al., 2018). In the Akt/mTOR pathway, protein mTOR can be activated by Akt, which can inhibit cellular apoptosis and promote cell proliferation (Zhang et al., 2017).

Hence, the protein expression in the Akt/mTOR pathway was detected by western blotting. As a result, the expression of p-Akt and p-mTOR was remarkably downregulated after baicalin treatment (Figure 3C). To investigate whether the Akt signal was a key determinant for the antitumor activity of baicalin in lung cancer, we specifically enhanced Akt activation using a pharmacological activator SC79 (5 μM) and the Akt overexpression plasmid. As a result, when the Akt activator SC79 was added to baicalin-treated lung cancer cells, the decreased cell proliferation, increased cell cycle arrest, and increased apoptosis were significantly rescued (Figures 4A–C). Next, the Akt overexpression plasmid was used to transfect H1299 and H1650 cells, which was verified by western blotting (Figure 4D). This result showed that the antitumor activity of baicalin was remarkably attenuated by Akt overexpression, followed by upregulated phosphorylation of mTOR and expression of cell cycle-related proteins in the G1/S phase including CDK2, CDK4, and Cyclin E2. In conclusion, our results demonstrate that baicalin has antitumor potential via suppressing the Akt/mTOR pathway in vitro.

The antitumor effect of baicalin was also evaluated in vivo. The establishment of the subcutaneous tumor model and drug administration regimen in nude mice is shown in Figure 5A. First, BALB/c nude mice were used to establish the subcutaneous tumor model. When xenografts reach 100 mm³, the mice were, respectively, divided into the control group (DMSO), baicalin group (20 mg/kg), SC79 group, and SC79 combined with baicalin group. The data showed that tumor volume was significantly inhibited by baicalin treatment when compared with the control group (Figure 5B). Importantly, there was no significant change in the body weights in four different groups, which indicated that there was no or much lower toxicity when treated with baicalin (Figure 5C).

Then, the apoptotic and cell cycle arrest-related proteins from different treatment groups were detected by immunohistochemical staining. As shown in Figure 6A, baicalin treatment caused a significant decrease of p-Akt and p-mTOR as well as proteins CDK4 and Cyclin E2. Importantly, these results were also demonstrated by western blotting (Figure 6B). In the further study, we found that co-treatment with baicalin and Akt activator SC79 significantly attenuated the decreased tumor size and upregulated the expression of p-Akt, p-mTOR, CDK4, and Cyclin E2, when compared with baicalin treatment alone (Figures 5, 6). These results suggest that baicalin could inhibit tumor growth in vivo by suppressing cell cycle progression via inhibiting the Akt signal pathway.