Sparteine exerts anticancer effect on human cervical cancer cells via induction of apoptosis, G0/G1 cell cycle arrest and inhibition of VEGFR2 signalling pathway

Purpose: To investigate the anticancer effects of sparteine against human cervical cancer. Methods: Cell viability was determined by CCK8 assay, while 4′, 6-diamidino-2-phenylindole (DAPI) staining was used for determination of apoptosis. Cell cycle analysis was done with flow cytometry, while cell invasion was monitored using Transwell invasion assays. Protein expressions were determined using Western blotting. Results: The results revealed that sparteine inhibited the viability of cervical cancer cells with halfmaximal inhibitory concentration (IC50) ranging from 10 to 25 μM. Sparteine exerted more profound antiproliferative effects on DoTc2 cells, with IC50 of 10 μM. However, minimal cytotoxicity was observed in normal cervical cells, as evident from the IC50 of 80 μM. Sparteine triggered the generation of ROS and apoptotic cell death in DoTc2 cells. The induction of apoptosis was accompanied by upregulation of Bax expression and downregulation of Bcl-2 expression. Sparteine caused arrest of DoTc2 cells at the G0/G1 phase of the cell cycle, and suppressed the expressions of cyclin A and cyclin B1. Transwell assay data showed that sparteine decreased the invasion ability of DoTc2 cells. Sparteine also inhibited the phosphorylation of VGFR2 in a concentration-dependent manner. Conclusion: Sparteine exhibits significant anticancer activity and may prove beneficial in cervical cancer chemotherapy.


INTRODUCTION
Cervical cancer imposes huge disease burden on all nations across the globe. Approximately, 260,000 cervical cancer mortalities and 530,000 new cases of cervical cancer were reported in 2012 alone [1]. Owning to disproportionate incidence of cervical cancer in low and high-income nations, it is often referred to as the 'disease of disparity' [2]. Cervical cancer is ranked the fourth major type of cancer in the world [3]. The treatments for cervical cancer involve radical hysterectomy, chemotherapy and/or radiotherapy [4].
However, the currently available chemotherapeutic agents have adverse effects on the quality of life of patients [5]. This has fuelled the search for novel and effective anticancer agents for combating this disease. One of the major concerns of researchers is identification of natural anticancer principles for the development of chemotherapy for cervical cancer [6]. Among natural products, alkaloids constitute a large and diverse group of metabolites in the plant kingdom. The potential of alkaloids as anticancer agents has increased tremendously with the discovery of the alkaloidal anticancer agents vinblastine and vincristine [7].
Sparteine is an important alkaloid isolated from Lupinus species. It belongs to the group of quinolizidine alkaloids and has been reported to exhibit analgesic activity [8]. Nonetheless, there is no single report in the literature on the anticancer activity of sparteine. Therefore, this study was undertaken to investigate the anticancer effects of sparteine against a panel of human cervical cancer cell lines and one normal cell line. Moreover, the effect of sparteine on the therapeutically important VEGFR2 pathway was investigated [9].

Determination of cell viability
CCK-8 assay was used for the determination of cell viability of DoTc2 cervical cancer cells. Transfected DoTc2 cells were seeded in 96-well plates and subjected to treatment with varied concentrations of sparteine at 37 ○ C for 24 h. Thereafter, 10 µL of CCK-8 reagent was added to the cell culture and incubated for 2 h at 37 ○ C in a humidified atmosphere containing 5 % CO 2 and 95 % O 2 . Optical density was read at 450 nm in a microplate reader, as an index of cell viability.

DAPI staining assay
Cervical cancer DoTc2 cells (0.6 × 10 6 ) were cultured in 6-well plates. They were treated with varied concentrations of sparteine, and incubated for 24 h at 37 ○ C. As the cells sloughed off, 10 μL cell culture was put onto a glass slide and subjected to staining with DAPI. The slides were covered with a cover slip and examined under a fluorescent microscope.

Transwell assay
The invasion abilities of the DoTc2 cells were determined with Transwell chamber invasion assay. The cells (1 × 10 4 ) were seeded in upper chamber of the transwell (8 μm pore size polycarbonate filters). This was followed by the incubation of the chambers at 37 °C for 24 h. The inserts were coated with extracellular matrix gel (50 μL) (ECM, Sigma, USA). Swabbing was performed to remove the non-invaded cells from the upper surface. However, the invaded cells on the lower surface were fixed with methanol and then stained with crystal violet (0.5%) for about 50 min. The cells were then washed with PBS, and finally counted under the light microscope (5 fields).

Cell cycle analysis
DoTc2 cells were treated with varied concentrations of sparteine and incubated for 24 at 37 ○ C. Thereafter, the cells were washed with phosphate buffered saline (PBS), and the KGN cells were stained with propidium Iodide (PI). The distribution of cells in cell cycle phases was assessed using FACS flow cytometer.

Western blot analysis
Sparteine-treated DoTc2 cells were harvested using centrifugation and then lysed in lysis buffer containing protease inhibitor. About 45 µg of protein from each sample was subjected to separation on 10 % SDS-PAGE. The protein bands were transferred to polyvinylidene difluoride (PVDF) membrane, and the membrane was then blocked with fat-free milk at room temperature for 1 h. The membrane was treated with appropriate primary antibodies at 4 ○ C overnight, and was subsequently incubated with secondary antibody. Finally, the signals were detected using Odyssey Infrared Imaging System (LI-COR, USA). Actin was used as control for normalisation.

Statistical analysis
All assays were performed in triplicate and the values are shown as mean ± standard deviation (SD, n = 3). Student's t-test was employed for statistical analysis using GraphPad Prism 7 software. Values of p < 0.05 were considered statistically significant.

Sparteine exerted growth inhibitory effects on cervical cancer cells
The effects of sparteine were evaluated against a panel of four cervical cancer cell lines (CaSki, DoTc2, SiHA, C-33A) and one normal cell line (HCvEpC) (Figure 1 A). The results indicated that sparteine suppressed the proliferation of all the cervical cancer cells, with IC 50 values ranging from 10 to 25 µM (Table 1). It was observed that sparteine exerted more significant anticancer effects on the DoTc2 cells, with an IC 50 of 10 µM (Figure 1 B). Evaluation of the anti-proliferative effects of sparteine on normal HCvEpC cells showed that sparteine exerted minimal growth inhibitory effects on these cells (IC 50 = 80 µM). The growth inhibitory effects of sparteine on the cervical cancer cells exhibited a dose-dependent pattern.

Sparteine promoted generation of ROS in DoTc2 cells
The effects of different concentrations of sparteine on ROS levels in DoTc2 cells were investigated. It was revealed that sparteine caused dose-dependent increases in the ROS levels of the DoTc2 cells ( Figure 2). The ROS level increased to 205 %, relative to 100 % in control. Moreover, the sparteine-induced increases in ROS levels were concentrationdependent.

Sparteine caused apoptotic cell death of DoTc2 cells
To ascertain if sparteine inhibited DoTc2 cell proliferation via induction of apoptotic cell death, DAPI staining of the sparteine-treated DoTc2 cells was performed. The results showed that sparteine caused nuclear fragmentation in DoTc2 cells in a dose-dependent manner (Figure 3 A). These alterations in the nuclear morphology of the DoTc2 cells were characteristic of apoptosis. Therefore, western blot analysis was performed to determine the expressions of Bax and Bcl-2 (Figure 3 B). The expression of Bax was found to increase while as that of bcl-2 decreased in the DoTc2 cells, upon sparteine treatment. The effect of sparteine on the distribution of the DoTc2 cells in different cell cycle phases was also studied. The results indicated that sparteine treatment caused a significant increase in the G 0 /G 1 phase of the cell cycle, which indicated G 0 /G 1 arrest (Figure 4 A). This was also accompanied by downregulation of cyclin A and cyclin B1 expressions in a dose-dependent manner (Figure 4 B).

Sparteine inhibited invasion of the DoTc2 cells
Transwell assay was used to monitor the effect of 10 µM (IC 50 ) of sparteine on the invasion of the DoTc2 cervical cancer cells. The results showed that sparteine caused significant decrease in the invasion of the DoTc2 cells ( Figure 5). Invasion of DoTc2 cervical cancer cells decreased to 38 %, when compared to 100 % in untreated control.

Sparteine deactivated VEGFR2 pathway in DoTc2 cells
The effect of sparteine was also investigated on the phosphorylation status of VEGFR2 in the DoTc2 cells ( Figure 6). The results showed that the phosphorylation of VEGFR2 cells decreased concentration-dependently upon treatment with sparteine. Figure 6: Effect of sparteine on the phosphorylation of p-VEGFR as depicted by western blot analysis. Experiments were performed in triplicate

DISCUSSION
Owing to drawbacks in cervical cancer chemotherapy, the development of new treatment strategies is of utmost priority for cancer researchers across the globe. Huge research efforts are regularly devoted to exploring effective treatments, as well as minimising the toxicities of the identified drugs [10]. Many of the anticancer drugs exhibit narrow therapeutic windows because of their low selectivity against cancer cells. Another goal of the researchers is to develop drugs that selectively target cancer cells or reduce their malignancy without any effect on normal cells [11].
One of the approaches is to explore chemical scaffolds isolated from terrestrial plants [12]. In the present study, the anticancer effect of the alkaloid sparteine against a panel of cervical cancer cells, was investigated. The results showed that sparteine selectively killed the cervical cancer cells, with greater profound effects on the DoTc2 cells. These observations are also supported by other studies carried on the quinolizidine alkaloids. For example, quinolizidine alkaloids from Sophora flavescens have been shown to inhibit the growth of cancer cells [13]. Studies have shown that many of the plant-derived alkaloids increase the generation of ROS in cancer cells [14]. For instance, it has been reported that the plant derived alkaloid berberine induced accretion of ROS in prostate cancer cells [15].
The effect of sparteine on ROS levels in the DoTc2 cells was also studied. Interestingly, it was revealed that sparteine caused accretion of significant amounts of ROS in DoTc2 cells.
Studies have shown that ROS generation is linked to induction of apoptosis in cancer cells [16]. Indeed, DAPI staining of the sparteinetreated DoTc2 cervical cancer cells revealed that sparteine caused nuclear fragmentation of the DoTc2 cells. The apoptosis was further confirmed with the expression patterns of Bax and Bcl-2 in the DoTc2 cells. It is known that Bax and Bcl-2 are important biomarker proteins of apoptosis [17]. The results showed that sparteine upregulated Bax and downregulated Bcl-2. Previous studies have shown that the alkaloid sanguinarine induced apoptosis in breast cancer cells [18]. Alkaloids have also been reported to cause cell cycle arrest in cancer cells [19]. Neferine, an alkaloid, has been shown to cause cell cycle arrest in lung cancer cells [19]. In the present study, it was found that sparteine caused arrest of the DoTc2 cells in the G 0 /G 1 phase of the cell cycle. Sparteine also inhibited invasion of the DoTc2 cells, indicating its anti-metastatic potential. Vascular endothelial growth factor-2 (VGFR2) is an important therapeutic target for cancer treatment [20]. The results obtained in this study showed that sparteine inhibited the phosphorylation of VGFR2. This is suggestive of the anti-cancer potential of sparteine.

CONCLUSION
The findings of this study show that sparteine exerts growth-inhibitory effects on DoTc2 cells via ROS-mediated apoptosis and G 0 /G 1 arrest. It also suppresses the invasion of DoTc2 cells via inhibition of VGFR2 phosphorylation. Therefore, sparteine is a potential lead molecule for the development of cervical cancer chemotherapy.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
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