A Study on the Effects of Matrine on Radiotherapy Sensitivity in Lung Cancer Based on the PAK6 and Wnt/β-Catenin Signaling Pathways

Abstract: Objective To investigate the effect of matrine on the radiosensitivity of lung cancer and its underlying mechanisms. Methods Human non-small cell lung cancer A549 cells were divided into a control group, a 2 Gy radiotherapy group, a 4 Gy radiotherapy group, a matrine group, a matrine plus 2 Gy group, a matrine plus 4 Gy group, a sodium glycididazole plus 2 Gy group, and a sodium glycididazole plus 4 Gy group. Clonogenic assay, cell proliferation assay, and apoptosis assay were used to evaluate the impact of matrine on the radiosensitivity of A549 cells. A subcutaneous xenograft tumor model in nude mice was established, with groups including a control group, a 5 Gy radiotherapy group, a matrine group, a matrine plus 5 Gy group, and a sodium glycididazole plus 5 Gy group; tumor volume was recorded. Western blotting was employed to detect the expression of β-catenin, p21-activated protein kinase 6 (PAK6), c-myc, and caspase-3 in A549 cells and mouse tumor tissues. Results Compared with the radiotherapy-alone group, the matrine-plus-radiotherapy groups showed significantly reduced clonogenic formation and cell proliferation in A549 cells ( P <0.001), and cell apoptosis increased significantly ( P <0.001), the tumor volume in nude mice was significantly reduced ( P <0.001), the expression of PAK6, c-myc, and phosphorylated β-catenin proteins was significantly downregulated in both cells and nude mouse tumor tissues ( P <0.05, 0.01, 0.001), and Caspase-3 protein expression was significantly upregulated ( P <0.001). Conclusion: Matrine enhances the radiosensitivity of lung cancer by disrupting PAK6 expression and inhibiting the Wnt/β-catenin signaling pathway.

Lung cancer is the leading cause of cancer-related mortality, with a 5-year survival rate of only 15%. Based on histological differences, it is classified into small-cell lung cancer and non-small-cell lung cancer; the latter is further subdivided into adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma. ^[1] Early-stage lung cancer often presents with subtle clinical symptoms, and most patients are diagnosed at intermediate or advanced stages, resulting in a poor prognosis. ^[2] Currently, the main treatment modalities for lung cancer are surgery and radiotherapy. Approximately 65% of patients with non-small cell lung cancer require radiotherapy to enhance therapeutic efficacy; however, prolonged radiotherapy can lead to radiation resistance and tissue damage, among other adverse effects. ^[3] Therefore, selecting a scientifically sound and rational pharmacological regimen in combination with radiotherapy to enhance the radiosensitivity of lung cancer is of paramount importance in its clinical management.

Matrine is derived from the leguminous plant Sophora flavescens. Sophora flavescens Alkaloids extracted from Alt. exhibit significant therapeutic efficacy against tumors. ^[4-6] Studies have shown that matrine not only inhibits tumor cell proliferation and promotes tumor cell apoptosis, but also enhances the radiosensitivity of cancers such as gastric cancer and breast cancer. ^[7-11] The effects and underlying mechanisms of matrine on radiotherapy for lung cancer remain unclear. This study aims to elucidate these effects and mechanisms, thereby providing a reference for the clinical management of lung cancer.

1   Materials and Instruments

1.1 Cell

The human non-small cell lung cancer A549 cells were purchased from the Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

1.2 Animal

1.3 Pharmaceuticals and Reagents

Matrine (batch no. 519-02-8, purity 98%) was purchased from Shanghai Mairui’er Chemical Technology Co., Ltd.; sodium glycididazole (batch no. 201205284, purity 95.0%) was purchased from Shandong Luye Pharmaceutical Co., Ltd.; DMEM culture medium and fetal bovine serum were purchased from Gibco, USA; the BCA protein quantification kit, MTT assay kit, and cell apoptosis assay kit were purchased from Shenyang Wanlei Biotechnology Co., Ltd.; antibodies against p21-activated protein kinase 6 (PAK6), caspase-3, c-myc, phosphorylated β-catenin, β-catenin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and goat anti-rabbit IgG were all purchased from CST, USA.

1.4 Instrument

Microplate reader (Thermo Fisher Scientific, USA); 23Ex medical high-energy electron linear accelerator (Varian, USA); flow cytometer (Beckman, USA); electron microscope (Nikon, Japan); electrophoresis apparatus (Shanghai Tianneng Technology Co., Ltd.).

2   Method

2.1 Cell culture

A549 cells stored in liquid nitrogen were rapidly thawed in a 37°C water bath and then cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO₂. ₂ Cells were cultured in a incubator, and passaged when confluence reached 80%. Cells in the logarithmic growth phase were then used for subsequent experiments.

2.2  Matrine on A549 Effects on cell proliferation

Take A549 cells in the logarithmic growth phase, at 4×10 ³ /Cells were seeded into a 96-well plate, with a control group and three groups treated with matrine at concentrations of 2, 4, and 8 μmol/L (matrine dissolved in DMEM culture medium). After the cells had adhered to the plate, the medium was replaced with DMEM containing the respective concentrations of matrine; the control group received drug-free medium. The cells were then cultured for 24, 48, and 72 hours, after which 20 μL of MTT solution was added to each well and incubated for 4 hours. Subsequently, 200 μL of DMSO was added, and the absorbance was measured at 490 nm using a microplate reader. A ).

2.3  Matrine combined with radiotherapy on A549 Effects on cell proliferation

A549 cells in the logarithmic growth phase were seeded at 4×10 ³ /Cells were seeded into a 96-well plate, with the following groups established: control, 2 Gy radiotherapy, 4 Gy radiotherapy, matrine (4 μmol/L), matrine (4 μmol/L) combined with 2 Gy, matrine (4 μmol/L) combined with 4 Gy, sodium glycididazole (25 μg/mL) combined with 2 Gy, and sodium glycididazole (25 μg/mL) combined with 4 Gy. Matrine and sodium glycididazole were dissolved in DMEM culture medium. After the cells had adhered to the plate, the control group was supplemented with drug-free medium, while all drug-treated groups were switched to DMEM containing the respective drugs. Following 2- or 4-Gy ionizing radiation in the radiotherapy groups, 20 μL of MTT solution was added to each well and incubated for 4 hours; subsequently, 200 μL of DMSO was added, and absorbance was measured at 490 nm using a microplate reader. A 。

Ionizing radiation conditions: a Varian 23EX medical high-energy electron linear accelerator from the United States, using 6 MV X-rays; during irradiation, the cell culture dish was covered with a 2-cm-thick acrylic plate, the source-to-dish distance was 100 cm, and the dose rate was 3.2 Gy/min.

2.4 Matrine combined with radiotherapy on A549 Effects on Cell Cloning Formation

A549 cells in the logarithmic growth phase were seeded at 3×10 ² Cells were seeded at a density of 1,000 cells per well in a 6-well plate, with the following experimental groups: control, 2 Gy radiation, 4 Gy radiation, matrine (4 μmol/L), matrine (4 μmol/L) combined with 2 Gy, matrine (4 μmol/L) combined with 4 Gy, sodium glycididazole (25 μg/mL) combined with 2 Gy, and sodium glycididazole (25 μg/mL) combined with 4 Gy. Matrine and sodium glycididazole were dissolved in DMEM culture medium; the control group received DMEM without any drug, while each drug-treated group received DMEM containing the respective drug. The radiation groups were exposed to X-rays at doses of 2 or 4 Gy. Cells were then cultured in a CO2 incubator for 10–20 days, with the culture medium replaced every 7 days. Culturing was terminated when macroscopically visible colonies appeared; the medium was discarded, cells were fixed in anhydrous ethanol for 30 minutes, followed by incubation with 1 mL of 0.01% crystal violet for 1 hour. After discarding the staining solution and washing, single-colony colony counts were performed under a microscope, and the colony-forming efficiency was calculated.

Colony-forming efficiency (PE) = Number of colonies / Number of seeded cells

2.5  Matrine combined with radiotherapy on A549 Effects of Cell Apoptosis

A549 cells in the logarithmic growth phase were seeded at 5×10 ⁵ /Cells were seeded in 6-well plates, with the following experimental groups: control group, 2 Gy radiotherapy group, 4 Gy radiotherapy group, matrine (4 μmol/L) group, matrine (4 μmol/L) combined with 2 Gy group, matrine (4 μmol/L) combined with 4 Gy group, sodium glycididazole (25 μg/mL) combined with 2 Gy group, and sodium glycididazole (25 μg/mL) combined with 4 Gy group. Matrine and sodium glycididazole were dissolved in DMEM culture medium; the control group received DMEM without any drugs, while each drug-treated group received DMEM containing the respective drug, and each radiotherapy group was exposed to X-ray irradiation at doses of 2 or 4 Gy. Following the instructions provided with the cell apoptosis assay kit, 5 × 10 ⁵ Collect the cells in a centrifuge tube, resuspend them in 500 μL of Binding Buffer, add 5 μL of Annexin V-FLTC and 5 μL of PI dye, mix thoroughly, and incubate in the dark at room temperature for 15 minutes. Set up single-positive controls for Annexin V-FLTC and PI to adjust fluorescence compensation, and a blank control to optimize instrument voltage settings. Use a flow cytometer to measure the fluorescence intensities of the samples, and analyze the apoptotic cell population using FlowJo v5.573 software.

2.6 Effects of Matrine Combined with Radiotherapy on a Subcutaneous Xenograft Tumor Model in Nude Mice

Based on preliminary experiments and literature reports ^[11-12] After one week of acclimatization in nude mice, the animals were randomly assigned to a control group, a 5 Gy radiotherapy group, a matrine (50 mg/kg) group, a matrine (50 mg/kg) plus 5 Gy group, and a sodium glycididazole (25 mg/kg) plus 5 Gy group, with five mice in each group. Log-phase A549 cells were collected, resuspended in PBS, and adjusted to a cell density of 1×10 ⁸ /mL; the left axilla of mice was disinfected with a 75% ethanol solution, and 100 μL of A549 cells was then administered by subcutaneous injection into the left axilla of nude mice. Matrine and sodium glycididazole were dissolved in physiological saline, and when the tumor volume in the nude mice reached 100 mm ³ During the treatment period, mice were administered 100 μL of the respective drug by intragastric gavage every 2 days, while the control group received an equal volume of saline by gavage; the radiotherapy group underwent radiotherapy once every 3 days. Tumor volume was measured and calculated every other day throughout the treatment. After 3 weeks of drug administration, the mice were euthanized by cervical dislocation, and tumor tissues were excised.

2.7   Matrine combined with radiotherapy on A549 In cells and tumor tissues PAK6 , Caspase-3 , c-myc , β-catenin Impact on protein expression

Transfer 50 mg of tumor tissue into a centrifuge tube, add 250 μL of RIPA protein lysis buffer and 2.5 μL of PMSF protease inhibitor, homogenize on ice, then centrifuge at 12,000×g. g Centrifuge at 4°C for 10 minutes, collect the supernatant—this is the total tissue protein. Take A549 cells treated with matrine in combination with radiotherapy, add 200 μL of RIPA lysis buffer and 2 μL of PMSF protease inhibitor, lyse on ice, then centrifuge at 12,000× g Centrifuge at 4°C for 10 minutes, then collect the supernatant, which represents the total cellular protein.

After quantification of BCA protein, samples were heated in a boiling water bath to induce denaturation, then separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat milk for 2 hours, followed by incubation overnight at 4°C with primary antibodies against PAK6, Caspase-3, c-Myc, phosphorylated β-catenin, β-catenin, and GAPDH (1:1000 dilution). After washing with TBST, secondary antibodies were added and the membrane was incubated at room temperature for 1.5 hours. Following another TBST wash, the luminescent substrate was prepared according to the ECL kit instructions, the PVDF membrane was immersed in the substrate for 0.5 minutes to allow reaction, and the membrane was exposed and photographed. The resulting images were analyzed using ImageJ software.

2.8  Statistical Analysis

Statistical analyses were performed using SPSS 19.0. All data are presented as mean ± standard deviation. Differences among multiple groups were assessed using one-way analysis of variance (ANOVA), while pairwise comparisons between two groups were conducted using the LSD method.

3   Result

3.1  Matrine on A549 Effects on cell proliferation

As shown in Figure 1, compared with the control group, treatment with matrine (2, 4, and 8 μmol/L) for 24, 48, and 72 hours resulted in a significant decrease in A549 cell viability ( P <0.001), indicating that matrine can effectively inhibit the proliferation of A549 cells. In conjunction with reported literature, ^[9] , the medium dose of 4 μmol/L matrine was selected for subsequent experiments.

3.2  Matrine combined with radiotherapy on A549 Effects on cell proliferation

As shown in Figure 2, compared with the control group, cell viability was significantly reduced in all groups except the 2 Gy radiotherapy group ( P <0.05, 0.01, 0.001); compared with the 2 Gy radiotherapy group, the cell viability was significantly reduced in the 2 Gy radiotherapy combined with matrine group and the 2 Gy radiotherapy combined with sodium glycididazole group ( P <0.001); compared with the 4 Gy radiotherapy group, the cell viability was significantly reduced in the 4 Gy radiotherapy combined with matrine group and the 4 Gy radiotherapy combined with sodium glycididazole group ( P <0.001); no significant difference in cell viability was observed between the matrine plus radiotherapy group and the sodium glycididazole plus radiotherapy group. These findings indicate that matrine can enhance the radiosensitivity of A549 cells, with an efficacy comparable to that of the radiosensitizer sodium glycididazole.

3.3 Matrine combined with radiotherapy on A549 Effects on Cell Cloning Formation

As shown in Figure 3, the cell colony-forming rates in the control group, the 2 Gy radiotherapy group, the 4 Gy radiotherapy group, the matrine group, the matrine plus 2 Gy group, the matrine plus 4 Gy group, the sodium glycididazole plus 2 Gy group, and the sodium glycididazole plus 4 Gy group were (59.36±2.29)%, (58.67±1.13)%, (34.06±2.30)%, (38.09±1.08)%, (26.33±0.87)%, (19.04±1.18)%, (24.42±3.19)% and (16.34±1.97)%, respectively. Compared with the control group, with the exception of the 2 Gy radiotherapy group, the cell colony-forming rates in all other groups were significantly reduced ( P <0.001); compared with the 2 Gy and 4 Gy radiotherapy groups, the cell clonogenic survival rates were significantly reduced in the matrine combined with radiotherapy group and the sodium glycididazole combined with radiotherapy group ( P <0.001); there was no significant difference in colony-forming efficiency between the matrine combined with radiotherapy group and the sodium glycididazole combined with radiotherapy group. These findings indicate that matrine can significantly enhance the in vitro radiosensitivity of A549 cells.

3.4 Matrine combined with radiotherapy on A549 Effects of Cell Apoptosis

As shown in Figure 4, the cell apoptosis rates in the control group, the 2 Gy radiotherapy group, the 4 Gy radiotherapy group, the matrine group, the matrine combined with 2 Gy group, the matrine combined with 4 Gy group, the sodium glycididazole combined with 2 Gy group, and the sodium glycididazole combined with 4 Gy group were (4.28±1.07)%, (4.07±0.43)%, (6.20±0.20)%, (6.11±1.34)%, (12.93±2.06)%, (29.33±1.15)%, (14.06±0.27)%, and (27.43±2.11)%, respectively. Compared with the control group, with the exception of the 2 Gy radiotherapy group, the cell apoptosis rates in all other groups were significantly increased ( P <0.05, 0.001); compared with the 2 Gy and 4 Gy radiotherapy groups, the cell apoptosis rates in the matrine combined with radiotherapy group and the sodium glycididazole combined with radiotherapy group were significantly increased ( P <0.001); there was no significant difference in the rate of cellular apoptosis between the matrine combined with radiotherapy group and the sodium glycididazole combined with radiotherapy group. These findings indicate that the combination of matrine and radiotherapy can significantly enhance the radiosensitivity of cells.

3.5 Effects of Matrine Combined with Radiotherapy on Subcutaneous Xenograft Tumors in Nude Mice

As shown in Figure 5, compared with the control group, tumor volume was significantly reduced in all groups of nude mice ( P <0.01, 0.001); compared with the 5 Gy radiotherapy group, both the 5 Gy plus matrine group and the 5 Gy plus sodium glycidazide group showed a significant reduction in tumor volume in nude mice ( P <0.001), indicating that matrine can significantly enhance the radiosensitivity of lung cancer in nude mice.

3.6 Matrine combined with radiotherapy on A549 In cells and tumor tissues PAK6 , Caspase-3 , c-myc , β-catenin Impact on protein expression

As shown in Figure 6, in A549 cells, with the exception of the 2 Gy radiotherapy group, in which only c-myc protein expression was significantly reduced compared with the control group ( P <0.001), while the expression of PAK6, c-myc, and phosphorylated β-catenin proteins was significantly reduced in all other groups ( P <0.05, 0.01, 0.001), and Caspase-3 protein expression was significantly increased ( P <0.01, 0.001); compared with the 2 Gy and 4 Gy radiotherapy groups, the matrine combined with radiotherapy group and the sodium glycididazole combined with radiotherapy group showed significant reductions in PAK6, c-myc, and phosphorylated β-catenin ( P <0.05, 0.01, 0.001), and Caspase-3 protein expression was significantly increased ( P (<0.01, 0.001), indicating that matrine can downregulate PAK6 expression in A549 cells, inhibit the Wnt/β-catenin signaling pathway, and promote cell apoptosis.

As shown in Figure 7, in nude mice bearing subcutaneously implanted tumors, the protein expression levels of PAK6, c-myc, and phosphorylated β-catenin were significantly reduced in all treatment groups compared with the control group ( P (<0.05, 0.01, 0.001), the caspase-3 protein expression was significantly increased in the matrine combined radiotherapy group and the sodium glycididazole combined radiotherapy group ( P <0.001); compared with the 5 Gy radiotherapy group, the matrine combined with radiotherapy group and the sodium glycididazole combined with radiotherapy group showed significant reductions in PAK6, c-myc, and phosphorylated β-catenin ( P <0.001), and Caspase-3 protein expression was significantly increased ( P <0.001), indicating that matrine can downregulate PAK6 expression in the subcutaneous xenograft model in nude mice and inhibit the Wnt/β-catenin signaling pathway.

4 Discussion

Lung cancer is the malignancy with the highest incidence and mortality worldwide, with surgery and radiotherapy serving as its primary treatment modalities. Although state-of-the-art three-dimensional conformal radiotherapy can significantly enhance therapeutic efficacy, the issue of radioresistance remains unresolved. ^[13] Increasing the frequency and dose of radiotherapy is the primary strategy for enhancing tumor radiosensitivity; however, the severe pulmonary toxicity it induces is detrimental to patient prognosis. ^[14] Traditional Chinese medicine complexes and individual TCM constituents exhibit favorable therapeutic efficacy with minimal adverse effects in the treatment of cancer; consequently, an increasing number of studies are focusing on the combination of TCM with radiotherapy for cancer therapy. Matrine, an alkaloid extracted from Sophora flavescens, a leguminous plant of the genus Sophora, effectively inhibits tumor proliferation and migration; however, its combined use with radiotherapy for lung cancer has yet to be thoroughly investigated. This study found that the combination of matrine and radiotherapy significantly outperformed radiotherapy alone in suppressing A549 cell proliferation and colony formation and in promoting apoptosis. Moreover, compared with radiotherapy alone, the matrine–radiotherapy combination markedly inhibited the growth of subcutaneous xenograft tumors in nude mice, suggesting that matrine can enhance the therapeutic efficacy of radiotherapy and increase tumor radiosensitivity.

PAK is a serine/threonine kinase that regulates processes such as maintenance of cell morphology and cytoskeletal support. ^[15] Studies have shown that PAK6 is highly expressed in cancers such as lung cancer, breast cancer, and prostate cancer, where it can promote tumorigenesis and tumor progression. ^[16-18] This study found that PAK6 protein expression in A549 cells was significantly reduced after radiotherapy; moreover, co-administration of matrine with radiotherapy resulted in an even more pronounced downregulation of PAK6 protein, suggesting that matrine can attenuate PAK6 expression in lung cancer cells, thereby enhancing their sensitivity to radiotherapy. Apoptosis is a complex, programmed cell death process regulated by multiple intracellular genes. Caspase-3 is a member of the caspase family and serves as an executioner protein in apoptosis; upon activation, Caspase-3 undergoes proteolytic cleavage, leading to increased expression and driving apoptosis into an irreversible phase. Studies have shown that interfering with PAK6 expression in tumor cells can promote the expression of the apoptotic protein Caspase-3. ^[19-20] This study found that matrine in combination with radiotherapy significantly increases Caspase-3 protein expression, suggesting that matrine may promote cell apoptosis by inhibiting PAK6 expression and thereby enhancing Caspase-3 expression. The Wnt/β-catenin signaling pathway is a canonical intracellular signaling cascade in tumor cells that regulates tumor growth and metastasis. Under pathological conditions, activation of the Wnt/β-catenin pathway leads to the formation of a complex between β-catenin and other protein molecules, which in turn modulates the expression of downstream target proteins and promotes tumorigenesis and tumor progression. ^[21] c-Myc is a downstream target gene of the Wnt signaling pathway, regulating the expression of both oncogenes and tumor suppressor genes. PAK family proteins are closely associated with the activation of the Wnt/β-catenin signaling pathway. ^[22-23] This study found that matrine combined with radiotherapy significantly inhibits the activation of the Wnt/β-catenin signaling pathway and its downstream target c-myc, compared with radiotherapy alone.

In summary, matrine enhances the radiosensitivity of lung cancer, likely through the suppression of PAK6 expression and the Wnt/β-catenin signaling pathway, thereby modulating tumor cell proliferation and apoptosis.

Source: Li Xiuwei, Wang Jinan, and Zhang Jian. “Study on the Effects of Matrine on Radiotherapy Sensitivity in Lung Cancer Based on the PAK6 and Wnt/β-catenin Signaling Pathways” [J]. Chinese Traditional Herbal Medicines, 2021, 52(2): 447–453.