World Journal of Traditional Chinese Medicine

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 6  |  Issue : 4  |  Page : 500--507

A study on the mechanism of bruceine D in the treatment of non-small cell lung cancer H1299 cells


Xiao-Yun Shen1, Chao-Yue Su2, Yan-Yan Yan3, Ling-Ling Zhang2, Qiao-Ru Guo2, Hu-Biao Chene4, Shuhela Zhumabieke5, Yelxat Danabek5, Jia-Jun Li2, Yun Liu2, Bolat Makabel6, Jian-Ye Zhang2,  
1 Guangdong Provincial Key Laboratory of Molecular Target and Clinical Pharmacology, School of Pharmaceutical Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou; Xinjiang Institute of Materia Medica; Institute of Traditional Chinese Medicine, Xinjiang Medical University, Urumqi, P. R. China
2 Guangdong Provincial Key Laboratory of Molecular Target and Clinical Pharmacology, School of Pharmaceutical Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, P. R. China
3 Guangdong Provincial Key Laboratory of Molecular Target and Clinical Pharmacology, School of Pharmaceutical Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou; Collaborative Innovation Center for Cancer, Institute of Respiratory and Occupational Diseases, Medical College, Shanxi Datong University, Datong, P. R. China
4 School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, P. R. China
5 Xinjiang Institute of Materia Medica, Urumqi, P. R. China
6 Guangdong Provincial Key Laboratory of Molecular Target and Clinical Pharmacology, School of Pharmaceutical Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou; Institute of Traditional Chinese Medicine, Xinjiang Medical University, Urumqi; Key Laboratory Ethnomedicine (Minzu University of China), Ministry of Education, Beijing, P. R. China

Correspondence Address:
Prof. Bolat Makabel
Xinjiang Medical University, Urumqi 830011
P. R. China
Prof. Jian-Ye Zhang
Guangzhou Medical University, Guangzhou 511436
P. R. China

Abstract

Background: Non-small cell lung cancer (NSCLC) is considered one of the leading causes of cancer-related death. Despite the availability of drugs for the treatment of NSCLC, the need for the development of novel agents with high efficiency and fewer adverse effects remains unmet. The natural compound bruceine D (BD) is widely recognized for its notable anti-inflammatory, antiparasitic, and hypoglycemic activities. However, it is unclear whether BD can be used as a novel agent for NSCLC treatment. Materials and Methods: MTT and colony formation assays were used to assess the antiproliferative effect of BD on NSCLC cells. Wound healing and transwell assays were performed to determine the effect of BD on the migration and invasion of H1299 cells, respectively. Western blotting assay was used to detect the expression levels of proteins. Results: We demonstrated that BD significantly inhibited the proliferation of H1299, A549, and H226 cells with respective IC50 values of 6.06 ± 0.52, 7.15 ± 0.90, and 7.21 ± 0.75 μM. In addition, BD suppressed colony formation of H1299 cells in a dose-dependent manner. Following treatment with BD, the migration and invasive capabilities of H1299 cells were significantly inhibited in a dose- and time-dependent manner. Moreover, the results of Western blotting demonstrated that BD treatment resulted in the upregulation of the protein expression of E-cadherin and downregulation of the expression of N-cadherin, twist, snail, integrin αv, integrin β4, matrix metalloproteinase-7, and β-catenin proteins. Conclusion: BD inhibits proliferation, migration, and invasion of NSCLC cells; therefore, BD may be considered for its potential in adjuvant therapy for NSCLC.



How to cite this article:
Shen XY, Su CY, Yan YY, Zhang LL, Guo QR, Chene HB, Zhumabieke S, Danabek Y, Li JJ, Liu Y, Makabel B, Zhang JY. A study on the mechanism of bruceine D in the treatment of non-small cell lung cancer H1299 cells.World J Tradit Chin Med 2020;6:500-507


How to cite this URL:
Shen XY, Su CY, Yan YY, Zhang LL, Guo QR, Chene HB, Zhumabieke S, Danabek Y, Li JJ, Liu Y, Makabel B, Zhang JY. A study on the mechanism of bruceine D in the treatment of non-small cell lung cancer H1299 cells. World J Tradit Chin Med [serial online] 2020 [cited 2021 Mar 4 ];6:500-507
Available from: https://www.wjtcm.net/text.asp?2020/6/4/500/303579


Full Text



 Introduction



Lung cancer is currently known as the most common form of cancer and the leading cause of cancer deaths worldwide.[1],[2] Two major types of lung cancer, namely non-small cell lung cancer (NSCLC) (85%) and small cell lung cancer (15%), have been noted.[3] Significant progress has been made in the treatment of NSCLC over the past decade.[4],[5],[6] Unfortunately, NSCLC is highly invasive, accounting for the high mortality rate in lung cancer patients.[7],[4] The overall survival rate of patients remains markedly low.[8] At present, chemotherapy, radiotherapy, and surgery remain the primary treatment approaches for lung cancer.[9],[10] However, radiotherapy and/or chemotherapy are often associated with toxic side effects.[9] Therefore, it is necessary to develop new anticancer drugs with higher efficiency and mild side effects.

Many natural compounds have been reported to exhibit potential antitumor activity.[11],[12] Bruceine D (BD) is a natural compound derived from the Chinese medicinal plant Brucea javanica[13] that exerts a variety of pharmacological activities, including hypoglycemic,[14] anti- inflammatory,[15] and anti-parasitic[16] activities. Our previous findings confirmed that BD has the ability to induce apoptosis in human chronic myeloid leukemia K562 cells.[17] However, little information is available to establish the potential antitumor efficacy of BD in NSCLC.

To this end, we explored the effects of BD on the proliferation, migration, and invasion of NSCLC cells. In this study, we observed significant inhibitory effects of BD on NSCLC cells. Treatment with BD also reduced H1299 cell migration and invasion in a dose- and time-dependent manner. There was a significant increase in E-cadherin protein levels after exposure to BD, while the expression levels of N-cadherin, twist, snail, integrin αv, integrin β4, matrix metalloproteinase-7 (MMP-7), and β-catenin were downregulated.

 Materials and Methods



Chemicals and cell culture

BD (≥98% pure) was purchased from the Chunqiu Biological Engineering Co., Ltd. (Nanjing, China). It was dissolved in dimethyl sulfoxide and stored at − 20°C as single-use aliquots. The NCI-H1299, A549, and H226 cell lines were purchased from the Shanghai Saibai Biotechnology Co., Ltd, Shanghai, and were validated by performing short tandem repeat analysis. All cell lines were cultured in RMPI 1640 medium (Gino Biomedical Technology Co., Ltd., Hangzhou) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin-streptomycin, followed by incubation at 37°C in a humidified atmosphere containing 5% CO2.

Cell proliferation assay

The MTT assay was performed based on the protocol established in a previous report.[18] Briefly, NSCLC cells were seeded in 96-well plates at a density of 3–5 ×103 cells/well. After 24 h, the cells were treated with different concentrations of BD. Twenty microliters of MTT solution was added per well after 68 h of incubation with BD. This was followed by incubating the cells in the dark at 37°C for 4 h. The absorbance of each sample was measured at 540 nm with 655 nm as a reference filter on a microplate reader (Bio-Rad Model 550, USA). The cytotoxicity of BD was evaluated by measuring IC50 values experimentally.

Colony formation assay

H1299 cells were seeded in 6-well plates at a density of 800 cells/well. The cells were then incubated with BD in progressively increasing concentrations (2.0, 4.0, 8.0, and 16.0 μM). After 2 weeks, cell colonies were fixed with 4% paraformaldehyde, followed by staining with 0.5% crystal violet. A number of colonies with more than 50 cells were counted under the microscope.[19]

Wound healing assay

A wound healing assay was performed to probe cell migration. Following treatment with different concentrations of BD, the monolayer formed by H1299 cells was scratched by a 200-μL yellow tip. Images were obtained at 0, 24, and 48 h after wounding.[13] Cell motility was quantified by measuring the percentage of area that healed after wounding. The cell mobility rate (%) was calculated with the following formula: percent mobility rate = (migrated cell surface area/total surface area) ×100.

Transwell assay

For the determination of cell invasion, we used 24-well transwell plates (8-mm pores) (Corning Costar, Corning, NY, USA). Briefly, 1 × 104 cells were seeded onto the upper chamber precoated with matrigel (Matrigel: basal medium 1:4). Following that, a 600-μL complete medium supplemented with 10% fetal bovine serum (FBS) was added to the lower chamber. After incubation in a constant temperature incubator for 24–48 h, the chambers were fixed in methanol for 2 h, stained with 0.5% crystal violet for 30 min, and photographed.[20]

Western blotting analysis

Western blotting was performed following the protocol established in a previous report.[21] Briefly, BD-treated cells were harvested and lysed in a lysis buffer supplemented with protease inhibitor cocktail and phenylmethylsulfonyl fluoride, followed by incubation on ice for 30min. After centrifugation at 14,000 g for 30 min at 4°C, the supernatant was collected and the protein concentration was determined by performing bicinchoninic acid protein assay using BCA Protein Assay Kit (Thermo Fisher Scientific, USA). An equal amount of protein samples were electrophoresed with 8%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to 0.45 μm polyvinylidene difluoride membranes (Millipore, USA). Following that, the membranes were blocked with 5% nonfat milk for 1h and subsequently incubated overnight at 4°C with specific primary antibodies. Then, the membranes were washed with Tris-Buffer Saline and Tween 20 (TBST) 3 times every 5 min, followed by incubation with horseradish peroxidase-conjugated anti-mouse/rabbit immunoglobulin G secondary antibody for 1 h at room temperature. Blots were visualized by Western Lightning® Plus-ELC kit (PerkinElmer, USA) and detected by ChemiDoc™ XRS+ (Bio-Rad, USA) [Table 1].{Table 1}

Statistical analysis

All experiments were performed in triplicates unless stated otherwise. Statistical analysis was carried out using SPSS 16.0 software (International Business Machines Corporation, Armonk, New York, US). The data were tested for significance using Student's t-test or one-way ANOVA. All quantitative data were expressed as mean ± standard deviation. Differences between groups were considered statistically significant if P < 0.05.

 Results



Inhibition of the proliferation of non-small cell lung cancer cells by bruceine D

Primarily, MTT assay was used to verify the effect of BD [the chemical structure is shown in [Figure 1]a on the cell proliferation ability of different NSCLC cell lines. As shown in [Figure 1]b, [Figure c], [Figure 1]d, BD significantly inhibited the proliferation of H1299, A549, and H226 cells with respective IC50 values of 6.06 ± 0.52, 7.15 ± 0.90, and 7.21 ± 0.75 μM.{Figure 1}

Inhibition of H1299 cell colony formation by bruceine D

The effects of BD on the proliferation ability of H1299 cells were further evaluated with colony formation assay. As shown in [Figure 2]a and [Figure 2]b, BD inhibited colony formation of H1299 cells in a dose-dependent manner. In conclusion, the aforementioned results indicated that treatment with BD could lead to the inhibition of the proliferation of NSCLC cells.{Figure 2}

Inhibition of the H1299 cell migration with bruceine D

The wound healing assay was performed to determine whether BD inhibited NSCLC cell migration. Consequently, the effect of BD was evaluated on H1299 cell migration. The H1299 cells were exposed to BD for 24 h with concentrations of 2.0, 4.0, and 8.0 μM. The mobility ratios calculated with respect to control were 56.25 ± 3.42%, 43.23 ± 3.86%, 29.59 ± 3.7%, and 18.83 ± 0.91% for respective BD concentrations. When the exposure to BD was extended to 48 h, the mobility ratios calculated with respect to control were 64.91 ± 2.54%, 51.53 ± 4.00%, 33.03 ± 1.21%, and 25.49 ± 1.32%, respectively [Figure 3]a and [Figure 3]b.{Figure 3}

Inhibition of the H1299 cellular invasion by bruceine D

The transwell assay was used to further study the ability of BD to inhibit H1299 cellular invasion. Cells were seeded onto upper chambers presoaked with matrigel, followed by incubation with 2.0, 4.0, 8.0, and 16.0 μM BD for 48 h. As shown in [Figure 4]a and [Figure 4]b, the number of H1299 cells invaded decreased in a dose-dependent manner with an increase in BD concentration. Consistently, after incubation with BD for 0, 24, or 48 h at 4.0 μM, the invasive ability of H1299 cells was significantly inhibited [Figure 4]c and [Figure 4]d. In a nutshell, the results described above indicated that BD inhibited the migration and invasive capacities of H1299 cells in a dose- and time-dependent manner.{Figure 4}

Inhibition of H1299 cellular migration and invasion by bruceine D through regulation of epithelial–mesenchymal transition and metastasis-related signaling pathways

Accumulated evidence has demonstrated that the activation of the epithelial–mesenchymal transition (EMT) signaling pathway is closely related to cancer metastasis. Therefore, we investigated whether BD exerted the antitumor effect by regulating the EMT-related signaling pathway. As shown in [Figure 5]a, expression levels of E-cadherin increased in a dose-dependent manner with correspondingly increasing BD concentrations, while the expression levels of twist, snail, and N-cadherin proteins notably decreased in H1299 cells. In addition, the integrin family plays an important role in cancer metastasis. Therefore, we studied changes in the expression levels of integrin αv and integrin β4 proteins in H1299 cells posttreatment with BD. The data showed that the expression levels of integrin αv and integrin β4 were significantly downregulated when exposed to BD [Figure 5]b. Given that previous studies have demonstrated the promotion of liver cell metastasis through the activation of the β-catenin/MMP-7 signaling pathway, we investigated further the effect of BD on β-catenin and MMP-7 protein expressions in H1299 cells. In consistence with the previously published data, β-catenin and MMP-7 were inhibited by BD in a dose-dependent manner [Figure 5]c.{Figure 5}

 Discussion



Despite significant progress in the diagnosis and treatment of NSCLC, the low 5-year overall survival rate in patients with advanced metastatic NSCLC and poor prognosis cannot be ignored.[4],[22] Certain limitations of the traditional approach of administering chemotherapy in the treatment of NSCLC have been noted owing to the extreme side effects and drug resistance.[23] Accumulated research has shown that certain natural plants contain multiple active compounds that exhibit potent antitumor activity, and more importantly, their administration leads to lower toxic side effects.[24] BD, a natural compound derived from the Chinese medicinal plant B. javanica, has demonstrated notable antitumor activity against a variety of tumors.[17],[25],[26] In this study, we demonstrated the inhibition of NSCLC tumor growth and suppression of H1299 cellular migration and invasion by treatment with BD. While elucidating the mechanism of the effects of BD, it was observed that the tumor-suppressive effects of BD can be mainly attributed to the regulation of the expression of metastasis-related proteins and EMT-related signaling pathways.

Many studies have shown that BD exhibits antiproliferative effects against a variety of cancer types.[13],[17],[25] A study performed by Wang et al. indicated that the proliferation of osteosarcoma cells was inhibited after exposure to BD.[13] Moreover, Cheng et al.[26] observed that BD inhibited hepatocellular carcinoma growth by targeting β-catenin/jagged1 pathways. Our previous research demonstrated the inhibitory effects of BD on the growth of hematological tumors.[17] In this research, we evaluated the inhibitory activities of BD on the proliferation of different NSCLC cells through MTT assay. The results demonstrated significant inhibition of H1299, A549, and H226 cells by treatment with BD, with respective IC50 values of 6.06 ± 0.52, 7.15 ± 0.90, and 7.21 ± 0.75 μM. In addition, the colony formation assay indicated that BD inhibited the proliferation of H1299 cells in a dose-dependent manner.

Tumor metastasis is a process by which malignant tumor cells migrate from the primary site to distant organs through blood vessels and complete the colonization. The epithelial–mesenchymal plasticity of cancer cells is considered a primary characteristic required to complete the metastasis process.[27] Tumor cells undergoing EMT lose the integrity of epithelial cells and exhibit a mesenchymal cell phenotype, thus possessing strong invasion and migration properties.[28],[29] E-cadherin promotes cell–cell adhesion to maintain structural integrity.[30] Reduction or loss of E-cadherin expression is a key change in the EMT.[31] During the EMT, proteins with higher flexibility (such as N-cadherin) replace adhesion proteins that connect the epithelial cells, leading to cell separation and enhanced mobility.[32] In a study conducted on triple-negative breast cancer, researchers observed that BD increased E-cadherin expression in breast cancer cells in a dose-dependent manner. However, treatment with BD downregulates β-catenin and vimentin expression.[33] In addition, a previous study has demonstrated that BD inhibited the migration of osteosarcoma cells by inhibiting Janus kinase/signal transducer and activator of transcription 3 phosphorylation.[13] In this study, we demonstrated the ability of BD in inhibiting the EMT-related signaling pathway through the downregulation of N-cadherin, β-catenin, twist, snail proteins, and upregulation of E-cadherin. Previous studies have shown that the activation of the Wnt/β-catenin signaling pathway in hepatocellular carcinoma promotes invasion, migration, and EMT progress in cancer cells.[34] MMP-7 is a downstream target of this pathway. Our results demonstrated inhibition of the expression of MMP-7 and β-catenin proteins by treatment with BD. Integrin is a type of transmembrane glycoprotein that exists on cell membrane and mainly plays a role in facilitating adhesion between the cells and extracellular matrix as well as the signal transduction process.[35] Studies have demonstrated that the upregulation of integrin β1 promotes the adhesion and migration of NSCLC cells.[36] In addition, integrin αv and integrin β4 have been demonstrated to promote invasion and migration in multiple tumors.[37],[38],[39] As an example, Wang et al.[37] showed that integrin αv promoted the migration and invasion of gastric cancer cells. In hepatocellular carcinoma and pancreatic ductal carcinoma, the overexpression of integrin β4 was associated with increased cell invasiveness and EMT.[38],[39] Furthermore, the upregulation of integrin β4 might be an indicator of poor prognosis in patients.[39] Here, we observed that integrin αv and integrin β4 were downregulated in H1299 cells after BD treatment. These findings confirmed that BD suppressed H1299 cellular migration and invasion by regulating the expression of EMT-related and cell adhesion-related molecular proteins.

 Conclusion



The results of this study indicated that BD inhibited the proliferation of NSCLC cells. Moreover, BD inhibited H1299 cellular migration and invasion in a dose- or time-dependent manner. The antitumor effect of BD may be attributed to the regulation of the expression of EMT-related and metastasis-related proteins [Figure 6].{Figure 6}

Acknowledgment

This work was supported by the National Natural Science Foundation of China (81773888, U1903126, and 81902152) and Natural Science Foundation of Guangdong Province (2020A1515010605), Open Founding of Key Laboratory Ethnomedicine Ministry of Education (KLEM-KF2019Y03).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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