Anticancer Effects and Molecular Mechanisms of Gossypin on Phosphatidylinositol 3-Kinase/Protein Kinase B Inhibition in Human Nonsmall Cell Lung Cancer Cell Line,A549

Abstract

Nonsmall cell lung cancer (NSCLC) accounts for approximately 80%–85% of all lung cancer patients. Gossypin, a flavonoid contained in Hibiscus vitifolius, has been reported as a potential anticancer agent. However, the effect of gossypin on NSCLC has not been elucidated. Herein, we evaluated the anticancer effects and investigated the molecular mechanisms of gossypin in NSCLC cell line, A549. We treated A549 cells with gossypin at different concentrations and performed cell viability and colony formation assays. As a result, gossypin inhibited cell proliferation. phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is known to be involved in cancer cell growth. Additionally, p-PI3K/p-Akt reduces p21 expression, resulting in cell cycle progression. Therefore, the effects of gossypin on the expression of PI3K, Akt, and p21 were investigated via Western blot analysis. Gossypin inhibited p-PI3K/p-Akt and induced p21 expression. Also, gossypin induced the sub-G₁ phase, which exhibits DNA fragmentation, indicating apoptosis. To corroborate these results, apoptosis and the expression of caspase-3 and poly ADP-ribose polymerase (PARP) were evaluated in gossypin-treated cells. Results showed that gossypin cleaved caspase-3 and PARP to induce apoptosis. Our study findings indicate a possible therapeutic effect of gossypin in NSCLC.

Introduction

Cancer is one of the most dangerous diseases worldwide. Furthermore, cancer incidence and cancer-related mortality continue to increase worldwide owing to eating habits that favor salty and high-fat foods and rapid aging. Globally, lung cancer mortality is 18.4% and comprises a major portion of cancer-related deaths. ¹ This is because approximately 70% of patients exhibit no symptoms in the early stages of lung cancer; however, if symptoms occur later, patients have already metastasized or advanced lung cancer. ² Nonsmall cell lung cancer (NSCLC) is a type of lung cancer that includes adenocarcinoma, squamous epithelial cancer, and large cell cancer, accounting for approximately 80%–85% of lung cancer cases. ³ Treatment of lung cancer includes surgery, radiation therapy, and chemotherapy. However, chemotherapy has limitations in that it causes various side effects in normal tissues. ⁴ Therefore, the development of natural materials with anticancer activity and low side effects continues to gain importance, and research on various molecular mechanisms, such as suppression of growth and enhancement of apoptosis, is being actively conducted.^5-7

Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is a signaling pathway that regulates cell angiogenesis, growth, metabolism, proliferation, and survival. ⁸ PI3K is a plasma membrane-associated lipo-phosphorylation kinase and is composed of 3 subunits: p55, p85, and p110. ⁹ p55 and p85 are regulatory subunits, and p110 is a catalytic subunit. PI3K is activated by extracellular excitation, such as cytokines, growth factors, and hormones. ¹⁰ Akt plays key roles in multiple cellular processes, including metabolism, transcription, migration, and proliferation. ¹¹ Akt is activated by phosphorylation of Ser473 or Thr308. p-Akt is involved in cell proliferation, motility, and inhibition of apoptosis. ¹² In addition, cell cycle arrest in the G₁ phase is inhibited by p-Akt. ¹³ In NSCLC, the PI3K/Akt pathway is closely related to tumor formation and disease progression, and increased activity of PI3K/Akt induces cell growth, inhibition of apoptosis, continuous angiogenesis, tissue invasion, and metastasis, indicating that the PI3K/Akt pathway may be targeted for chemotherapy of NSCLC. ¹⁴

A flavone in Hibiscus vitifolius, gossypin (3,5,8,3′,4′-pentahydroxy-7-O-glucosyl flavone), has been prescribed as a treatment for diabetes and inflammation.^15-17 Gossypin has been reported to inhibit cell proliferation in cancers such as that of the stomach, colon, and prostate.^18-20 In addition, gossypin has been demonstrated to significantly reduce the proliferation of melanoma tumors by regulating mitogen-activated protein kinase, cyclin D1, and Chk1 in vivo, and exhibits anticancer activity by inducing apoptosis through poly ADP-ribose polymerase (PARP) regulation. ²¹ Thus, gossypin could be used as a potential anticancer agent.

However, the effect of gossypin on NSCLC has not been elucidated. Therefore, we attempted to determine whether gossypin exerts an anticancer effect on NSCLC. In this study, we demonstrated that gossypin inhibited the PI3K/Akt pathway in A549 cells (NSCLC cells), thereby inhibiting proliferation and inducing apoptosis of A549. These results indicated the possibility that gossypin could be used as a novel anticancer agent for the treatment of NSCLC.

Materials and Methods

Cell Culture

The human NSCLC cell line A549 was purchased from the Korea Cell Line Bank. A549 cells were passaged in Dulbecco's Modified Eagle's Medium (HyClone) with 10% fetal bovine serum (HyClone), 1% penicillin, and streptomycin (HyClone). A549 cells were passaged every 2 to 3 days at 37 °C under 5% CO₂.

Cell Viability Assay

To investigate the effect of gossypin on cell viability, A549 cells were seeded into a 96-well plate (4 × 10³ cells/200 μL/well). Next, the cells were inoculated with gossypin (0, 20, 40, 60, 80, and 100 μM) and incubated for either 48 or 72 hr. The cell viability was measured by CellTiter96® Aqueous One Solution Assay of Cell Production (Promega) and a microplate reader (Sunrise™ BASIC; Tecan).

Colony Formation Assay

To examine the effect of gossypin on cell viability, A549 cells were seeded in a 24-well plate (1 × 10³ cells/mL/well). Next, the cells were treated with gossypin (0, 20, 40, 60, 80, and 100 μM) and incubated for 72 hr. Then the medium was changed to a new medium containing gossypin every 3 days and incubated for 14 days. A549 cells were washed once with phosphate-buffered saline (PBS), stained with a 1:1 mixture of 6% glutaraldehyde and 5% crystal violet for 30 min, and then washed thrice with PBS for 15 min each time. Stained cells were scanned using an Epson Perfection V37 scanner (Epson). Next, after extracting the crystal violet from the stained cell using 1 mL of 1% acetic acid aqueous solution, the absorbance was measured at 590 nm using a microplate reader (Sunrise™ BASIC; Tecan).

Cell Cycle Analysis

To examine the effect of gossypin on the cell cycle, A549 cells were seeded into a 24-well plate (1 × 10⁵ cells/mL/well). Next, the cells were treated with gossypin (0, 80, and 100 μM) and incubated for 72 hr. Then, the cells were stained using the Muse® Cell Cycle Kit (Merck Millipore) and analyzed using the Muse® Cell Analyzer (Merck Millipore).

Apoptosis assay

To investigate the effect of gossypin on apoptosis, A549 cells were seeded in a 24-well plate (2 × 10⁵ cells/mL/well). Next, A549 cells were treated with gossypin (0, 80, and 100 μM) and incubated for 72 hr. The cells were then stained with Muse® Annexin V & Dead Cell Assay Kit (Merck Millipore) and analyzed using the Muse® Cell Analyzer (Merck Millipore).

Western Blotting

The mechanism by which gossypin inhibits proliferation and induces apoptosis of A549 cells was investigated. A549 cells were seeded into a 6-well plate (2 × 10⁵ cells/3 mL/well). Then, the cells were treated with gossypin (0, 80, and 100 μM) and incubated for 72 hr. Next, the cells were washed with PBS, and cell lysis buffer was added to obtain cell lysate. Protein concentrations were determined by the Bio-Rad Protein Assay Kit (Bio-Rad), a product that standardizes bovine serum albumin. In brief, after adjusting the protein concentration to be the same, 15 μg/20 μL, we added a 2× sodium dodecyl sulfate (SDS) sample buffer and boiled it at 95 °C for 5 min. A 10% SDS polyacrylamide gel was prepared using the Mini-PROTEAN Electrophoresis System (Bio-Rad), loaded, and electrophoresis was performed with 14 μg of each sample in each well. The protein bands were then transferred to an Immobilon® transfer membrane (0.45 μm; Merck Millipore). The membrane was blocked at 20 °C to 25 °C for 1 hr with a 1× blocking buffer (BIOFACTORY). Then, primary antibodies, including β actin (#66009-1-Ig 1:10000, Proteintech), p-PI3K (#17366, 1:1500, Cell Signaling), PI3K (#4257, 1:1500, Cell Signaling), p-Akt (#9271, 1:1500, Cell Signaling), Akt (#9272, 1:1500, Cell Signaling), p21 (#sc-817, 1:400, Santa Cruz), cleaved caspase-3 (#9661, 1:1000, Cell Signaling), caspase-3 (#9662, 1:1000, Cell Signaling), PARP and cleaved PARP (#9544, 1:1000, Cell Signaling) were used to treat the membrane for 12 hr at 4 °C. Tris-buffered saline containing 0.1% Tween-20 (TBST, pH 8.0) was used to wash the membrane and then incubated with a secondary antibody (rabbit IgG, mouse IgG, horseradish peroxide-linked antibody, 1:2000, Cell Signaling) at room temperature for 90 min. The membrane was rinsed with TBST and treated with enhanced chemiluminescence solution (Thermo Scientific) as a substrate. The resulting bands were visualized using the Fusion Solo System (Vilber). The intensity of the bands was measured using ImageJ software (version 1.52; ImageJ National Institutes of Health).

Statistical Analysis

The data were analyzed using GraphPad Prism software (version 5.0; GraphPad) and marked as the mean ± standard error of the mean (SEM). The statistical significance between each group in the cell viability and colony formation experiments was revealed using a 1-way analysis of variance with Dunnett's test. The statistical significance between each group in the cell cycle analysis and apoptosis assay experiments was revealed using a 2-way analysis of variance with the Bonferroni test. A P value <.05 indicated a statistically significant difference.

Results

Gossypin Inhibits Proliferation of A549 Cells

Figure 1A depicts the chemical structure of gossypin. We treated A549 cells with gossypin for either 48 hr or 72 hr and determined cell viability by 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cell viability was not significantly reduced in A549 cells treated with gossypin for 48 hr (Supplemental Figure 1). However, upon treating cells with gossypin for 72 hr, cell viability gradually reduced from 60 μM (Figure 1B). We also observed that gossypin gradually inhibited colony formation from 40 μM (Figure 1C), thereby corroborating the above results. Therefore, gossypin inhibits cell viability and colony formation in A549 cells.

Figure 1.

Gossypin inhibits proliferation of A549 cells. (A) Chemical structure of gossypin. (B) A549 cells were exposed to different concentrations of gossypin for 72 hr, and cell viability was determined by MTT assay. (C) A549 cells were treated with the indicated concentrations of gossypin for 14 days. Colonies were stained using crystal violet, and photographs were captured. Crystal violet was extracted from the stained A549 cells to assess colony formation. The data are reported as the mean ± SEM. Statistically significant differences are based on comparison with the control (0 μM gossypin) using 1-way analysis with Dunnett's test (**P < .005, ***P < .001).

Gossypin Inhibits the PI3K/Akt Pathway and Induces p21 Expression to Inhibit Proliferation of A549 Cells

Since gossypin was evaluated to inhibit the proliferation of A549 cells, we determined whether gossypin regulates the protein expression of PI3K, Akt, and p21. A549 cells were treated with gossypin (0, 80, and 100 μM). At a concentration of 100 μM, gossypin reduced p-PI3K/p-Akt and induced the expression of p21 (Figure 2A). p21 is associated with cell cycle arrest.^22,23 Therefore, the effect of gossypin on the cell cycle was performed by cell cycle analysis. Gossypin, at a concentration of 100 μΜ, induced the sub-G₁ phase (Figure 2B). Collectively, these results showed that, in A549 cells, gossypin inhibits p-PI3K/p-Akt and induces the expression of p21, suggesting that it inhibits cell proliferation.

Figure 2.

Gossypin inhibits the proliferation of A549 cells by inhibiting the PI3K/Akt pathway and inducing the expression of p21. (A) A549 cells were treated with the indicated concentrations of gossypin for 72 hr. β-Actin is indicated as a loading control. Relative levels for Western blot bands were calculated by dividing the intensity of target protein bands by the density of the bands of Akt, PI3K, or β-actin protein. (B) A549 cells were treated with the indicated concentrations of gossypin for 72 hr, and cell cycle analysis was performed by cell cycle analysis. The data are reported as the mean ± SEM. Statistically significant differences are based on comparison with the control (0 μM gossypin) using a 2-way analysis of variance with the Bonferroni test (*P < .05).

Gossypin Induces Apoptosis of A549 Cells by Cleaving Caspase-3 and PARP

As elucidated by the above results, gossypin increases the sub-G₁ phase in A549 cells. Therefore, apoptosis in A549 cells treated with gossypin was determined by apoptosis assay. As a result, late apoptosis (7.68% ± 1.19%) and total apoptosis (10.57% ± 2.23%) were significantly increased in A549 cells treated with 100 μM gossypin (Figure 3A). Since cleaved caspase-3 induces apoptosis by cleaving PARP, ²⁴ we investigated the expression of caspase-3 and PARP to support the above results. Cleaved caspase-3 and cleaved PARP were observed to be increased in cells treated with gossypin (Figure 3B). This indicated that gossypin induced apoptosis of A549 cells by cleaving caspase-3 and PARP.

Figure 3.

Gossypin induces apoptosis of A549 cells by cl-caspase-3 and cl-PARP. (A) A549 cells were exposed to the indicated concentration of gossypin for 72 hr. Apoptosis was determined by apoptosis assay. (B) A549 cells were treated with the indicated concentration of gossypin for 72 hr. Relative levels for Western bands were computed by dividing the density of target protein bands by the density of the bands of PARP or caspase-3 protein. The data are reported as the mean ± SEM. Statistically significant differences are based on comparison with the control (0 μM gossypin) using a 2-way analysis of variance with the Bonferroni test (*P < .05).

Discussion

Lung cancer is one of the leading causes of all cancer-related deaths, with NSCLC accounting for 85% of them. ³ The negative prognosis of lung cancer is because of late diagnosis due to a lack of symptoms during early disease progression. ² Lung cancer treatment includes surgery, radiation, and chemotherapy. However, chemotherapy has limitations in that it exhibits various side effects. Therefore, we investigated the anticancer effects of gossypin, a natural product with fewer side effects, on NSCLC. ²⁵ Gossypin is a flavonoid, and various flavonoids show anticancer effects in NSCLC. ²⁶ The anticancer effect of gossypin on cancers, such as gastric, breast, and prostate cancer, has been validated in previous studies.^19,20,27 However, to date, the effect of gossypin on NSCLC has not been evaluated. In this study, we explored the anticancer effect of gossypin and the underlying molecular mechanisms on A549 cells.

The PI3K/Akt pathway promotes cell proliferation, cell survival, metabolism, angiogenesis, cell growth, and metastasis. ⁸ The phosphorylation of abnormal PI3K and Akt is involved in cell growth in various cancers ²⁸ ; phosphorylated PI3K and Akt reduce the expression of p21, thereby enabling the cell cycle to proceed.^13,29,30 In addition, phosphorylated PI3K/Akt, which is associated with tumor formation and progression, is overexpressed in NSCLC. ¹⁴ In this study, gossypin suppressed the proliferation of A549 cells. In detail, gossypin induced p21 expression by inhibiting the PI3K/Akt pathway, which is related to cell growth. Similar to the results of our study, apigenin, a flavone-like gossypin, showed anticancer effects by inhibiting Akt, and gossypin extracted from Vietnamese coriander inhibited Akt and showed anticancer effects in oral cancer.^31,32

Cancer causes cells to divide, undermining their ability to regulate the cell cycle. ³³ Gossypin has been reported to enhance cell cycle arrest in malignant gliomas, gastric cancer, and oral cancer.^21,31,34 Therefore, the effects of gossypin on the cell cycle of A549 cells were evaluated. In this study, the sub-G₁ phase was induced by gossypin. Cells in the sub-G₁ phase are the cells from which DNA fragments are generated, and they indicate that apoptosis is induced.^35,36 Through an apoptosis assay, we revealed that apoptosis was increased by the gossypin treatment. Subsequently, the expression was examined of caspase-3 and PARP, which are proteins related to apoptosis. PARP is related to DNA repair, and cleavage of PARP by cleaved caspase-3 interferes with DNA repair.^37,38 This leads to apoptosis as DNA cannot be repaired by cleaved caspase-3 and cleaved PARP. ³⁹ Our results showed that gossypin induced the expression of cleaved caspase-3 and cleaved PARP. This is consistent with the results of a previous study in which gossypin induced apoptosis in gastric cancer. ²⁰

In conclusion, our study demonstrated that gossypin inhibits the PI3K/Akt pathway in A549 cells, induces p21 expression, thereby causing cell cycle arrest, and cleaves caspase-3/PARP to induce apoptosis. Verifying the anticancer effects of gossypin in vivo is necessary, and if future studies show promising results, gossypin may be useful for treating NSCLC.

Supplemental Material

sj-tif-1-npx-10.1177_1934578X231194420 - Supplemental material for Anticancer Effects and Molecular Mechanisms of Gossypin on Phosphatidylinositol 3-Kinase/Protein Kinase B Inhibition in Human Nonsmall Cell Lung Cancer Cell Line, A549

Supplemental material, sj-tif-1-npx-10.1177_1934578X231194420 for Anticancer Effects and Molecular Mechanisms of Gossypin on Phosphatidylinositol 3-Kinase/Protein Kinase B Inhibition in Human Nonsmall Cell Lung Cancer Cell Line, A549 by Seong Uk Lee and Yoon Hee Kim in Natural Product Communications