Antitumor activity of gambogic acid lysinate in human SiHa cells in vitro and in vivo

doi:10.21203/rs.3.rs-1024268/v1

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Antitumor activity of gambogic acid lysinate in human SiHa cells in vitro and in vivo

https://doi.org/10.21203/rs.3.rs-1024268/v1

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Background: Cervical cancer is a major cause of death for women worldwide and human papillomavirus (HPV) infection is the main cause of cervical cancer. The purpose of this study was to explore the anti-tumor activity of gambogic acid lysinate and clarify its mechanism in SiHa cells.

Methods: In the present study, cell viability was detected by means of an MTT assay, a cell growth curve was drawn with Microsoft Excel 2010, the cell cycle and cell apoptosis were evaluated by flow cytometry, Western blotting was employed to explore the mechanism of gambogic acid lysinate, and caspase-3 activity was determined with a colorimetric Caspase-3 assay kit. Additionally, the in vivo antitumor activity of gambogic acid lysinate was studied through a xenograft tumor model established with nude mice.

Results: The results showed that gambogic acid lysinate inhibited the proliferation of both SiHa cells (half-maximal inhibitory concentration (IC₅₀) values: 0.83 μmol/l and 0.77 μmol/l for 48 h and 72 h) and HeLa cells (IC₅₀ >2 μmol/l). In SiHa cells, gambogic acid lysinate (1 and 2 μmol/l) inhibited cell proliferation and 2 μmol/l gambogic acid lysinate induced cell apoptosis and decreased the number of S phase cells. Both 1 and 2 μmol/l gambogic acid lysinate increased the number of G0/G1 phase cells. The results of a Western blot assay demonstrated that P53 and P21 were involved in SiHa cell G0/G1 phase arrest and that Bcl-2 and BAX were involved in SiHa cell apoptosis. An in vivo study showed that the growth of SiHa cell xenograft tumors was inhibited by gambogic acid lysinate (2.5 mg/kg body weight), however, gambogic acid lysinate (2.5 mg/kg body weight) had no significant effect on mouse weight gain.

Conclusions: gambogic acid lysinate is a promising candidate for cervical cancer therapy.

Infectious Diseases

Geriatrics & Gerontology

gambogic acid lysinate

cervical cancer

cell cycle arrest

apoptosis

xenografts

Cervical cancer is a major cause of death for women worldwide and human papillomavirus (HPV) infection is the main cause of cervical cancer. Among more than 30 type HPVs, HPV-16, -18, and -33 are associated predominantly with severe cervical dysplasia, carcinoma in situ, and invasive cervical carcinoma [1]. It was reported that HPV 18 DNA had been found integrated into the cellular genome and was amplified in HeLa, while HPV-16 DNA was integrated into the genomes of the SiHa [2]. There are approximately 530,000 new cases and 275,000 deaths every year worldwide [3] and the cervical cancer mortality in developing and underdeveloped countries accounts for 90% of the global mortality and is 18 times higher than that in developed countries [4]. In 2012, in developed countries, cervical cancer was the 11th most common type of female malignant tumor and the ninth most common cause of female malignant tumor-related death; however, in developing and underdeveloped countries, cervical cancer was the second most common type of female malignant tumor and the third most common cause of female malignant tumor-related death [5]. It was estimated by GLOBOCAN that in China there were 61,691 new cervical cancer cases in 2012, which accounted for 12% of the global new cervical cancer cases. In addition, in China, 29,526 women died of cervical cancer in 2012, reaching 11% of the global deaths from cervical cancer [6, 7]. Therefore, it is necessary to find new compounds from traditional Chinese medicine to treat cervical cancer, especially in developing and underdeveloped countries.

Gambogic acid (GA) is a major extract from gamboge and a brownish to orange resin derived from Garcinia hanburyi [8]. The crude extract of gamboge has a long history in the treatment of tumors in China. In 2004, the State Food and Drug Administration of China approved GA for evaluation in clinical trials for cancer treatment. It was reported that GA could inhibit tumor growth, suppress inflammation, reduce oxidation reactions, kill bacteria, etc. [9]. GA can inhibit the proliferation of a variety of tumor cells [10, 11]. However, the water solubility of GA is poor, therefore, its clinical application is limited. Gambogic acid lysinate (GAL) is the salt of gambogic acid and lysine. It was reported that GAL could induce apoptosis in breast cancer [12]; however, the antitumor activity of GAL in cervical cancer is unknown. In the present study, the antitumor activity of GAL in cervical cancer was explored, and the mechanism was discussed.

Reagents and Antibodies. Lysine was obtained from Beijing Solarbio Science and Technology Co. (Beijing, China). Gambogic acid (98%) was obtained from Nanjing Jingzhu Biotechnology Ltd. Gambogic acid lysinate (GAL) was synthesized according to established methods in our department. The molecular weight is 755.4 Da [12]. 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) was purchased from Sigma Aldrich. A Pierce BCA protein assay kit was purchased from Thermo Fisher Scientific Inc. Primary antibodies against Bcl-2, Bax, CDK-2, P53 and P21 were purchased from Cell Signaling Technology. An anti-GAPDH antibody was purchased from Santa Cruz Technology. Secondary antibodies were also purchased from Cell Signaling Technology. Immobilon Western Chemiluminescent HRP substrate was bought from EMD Millipore.

Cell culture and a cytotoxicity Assay. The human cervical cancer SiHa and HeLa cell lines were obtained from the National Biomedical Medical Cell Resource Bank. Completed culture medium was composed of Dulbecco’s modified Eagle’s medium (DMEM), 100 units/ml penicillin/streptomycin and 10% fetal bovine serum (FBS; HyClone). Both the SiHa and HeLa cell lines were cultured in complete medium and incubated in a 95% air/5% CO₂ humidified atmosphere at 37°C. The inhibitory of GAL on proliferation was detected by an MTT assay. Briefly, HeLa cells and SiHa cells were seeded in 96-well plates at 4 × 10³ cells per well. After culturing overnight, different concentrations (0, 0.5, 1, 1.5 and 2 µmol/l) of GAL were added to each well. After incubating for 24 h, 48 h or 72 h, 20 µl of MTT solution (5 mg/ml) was added to each well. After 4 h of incubation, the medium was removed, and 150 µl of DMSO was added to each well. A Spectra Max 190 Absorbance Microplate Reader (Sunnyvale) was used to measure the optical density at 570 nm, and the half-maximal inhibitory concentration (IC₅₀) was calculated with GraphPad Prism 5.0 (GraphPad).

Cell growth curve. Five hundred SiHa or HeLa cells were seeded in each well of a 6-well plate in 10% FBS DMEM. Saline or GAL (0.5 or 1.0 µmol/L) was added to each well after an overnight incubation. Triplicate wells are harvested by trypsinization every day for 6 days and counted with a cytometer. Growth curves were generated using Microsoft Excel 2010.

Cell cycle and cell apoptosis assays. SiHa cells were plated in a 25 cm² flask and exposed to various concentrations of GAL (0, 1, and 2 𝜇mol/L) for 24 h or 48 h. For the apoptosis assay, cells were digested, washed and centrifuged. Then, 300 µl of binding buffer was added to the cells, which were a final density of 1 × 10⁶ cells/ml. Annexin V-FITC and propidium iodide (PI) (Thermo Fisher Scientific, Inc.) were applied to stain the cells for 15 min in the dark. Then, flow cytometry was performed with a FACSCalibur and Cell Quest software (version 5.1; BD Biosciences) to detect apoptosis. For the cell cycle assay, cells were digested, washed, and fixed in 70% ethanol, and RNase (5 mg/ml) was added to treat the cells for 30 min. Following the addition of 50 mmol/l PI, flow cytometry was performed with a FACSCalibur and Cell Quest software to evaluate the cell cycle.

Western Blot Analysis. Different concentrations of GAL (0, 0.5, 1, and 2 µmol/l) was added to SiHa cells for 24 h or 48 h. The cells were rinsed twice with cold PBS, and RIPA buffer supplemented with a protease and phosphatase inhibitor cocktail (Roche, Beijing, China) was applied to lyse the cells for 30 min on ice. The cell lysates were centrifuged, and the supernatant was collected. A BCA assay kit was used to determine the protein concentration. Equal amounts and volumes of protein (30 µg) were resolved by SDS–PAGE, and the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, USA). Then, the membranes were blocked with 5% nonfat skim milk and sequentially incubated with primary and secondary antibodies. Protein bands were developed using Immobilon Western Chemiluminescent HRP substrate with an enhanced ChemiImager 5500 chemiluminescence system (ProteinSimple). ImageJ software (version 1.0; National Institutes of Health) was applied to calculate the optical density of the bands, which were standardized to the band for GAPDH.

Caspase 3 Activity Assay. Caspase-3 activity was evaluated with a colorimetric caspase-3 assay kit (Beyotime Institute of Biotechnology). SiHa cells (5x10⁵) were inoculated into 75 cm² culture flasks and treated with GAL (0, 0.5, 1.0 and 2.0 µmol/l). After 24 or 48 h of treatment, the SiHa cells were collected and resuspended in 50 µl of lysis buffer. Follow-up experiments were performed according to the study by Chunlai Shi et al [13].

In vivo xenograft mouse model. Four-week-old female BALB/c nude mice (20-24 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing China) and maintained at the Experimental Animal Center of Beijing Bozhiyuan Biotechnology Co., Ltd. Nude mice were kept in a specific pathogen-free environment in which the temperature was 22°C and the humidity was 40–50%. Studies involving mice were conducted in compliance with the Guidelines for the Care and Use of Laboratory Animals of Beijing Bozhiyuan Biotechnology Co., Ltd. SiHa cells (1 × 10⁷cells per mouse) were inoculated into the left armpit of nude mice by subcutaneous injection. Nine days after the inoculation, the mice were treated with either saline (control group) or 2.5 mg/kg GAL, every other day (GAL treated group). The body weight and tumor volume of the mice were measured every 2 days. After 10 days of treatment, the mice were euthanized, and their tumors were weighed recorded and photographed.

Statistical analysis. All data are represented as the mean ± standard deviation. SPSS 17.0 statistical software was used to perform statistical analysis. Efficacy was compared by one-way ANOVA or a two-sided unpaired Student’s t-test. When the p-value was less than 0.05, the difference was considered to be significant.

GAL inhibited the proliferation of SiHa and HeLa cervical cancer cells. The inhibitory effect of GAL on the proliferation of human cervical SiHa cells and HeLa cells was examined by an MTT assay. Cells were treated with different concentrations of GAL (0, 0.5, 1, 1.5 and 2 µmol/l) for 24, 48 or 72 h. The proliferative capacities of both SiHa cells and HeLa cells were decreased after treatment with GAL in a dose dependent manner. However, GAL had a stronger inhibitory effect on SiHa cells than HeLa cells (Figure 1). The IC₅₀ values of GAL for SiHa cells were 0.83 µmol/L at 48 h of treatment and 0.77 µmol/L at 72 h of treatment. However, the IC₅₀ value of GAL for HeLa cells was > 2 µmol/L. In addition, in a growth curve assay, the doubling speed of SiHa cells was significantly slower than that of HeLa cells. The inhibitory effect of GAL on SiHa cells was stronger than on HeLa cells (Figure 2).

Regulatory effects of GAL on the cell cycle and apoptosis in SiHa cells. In a cell cycle assay, 1 µmol/l GAL decreased the number of G2/M phase cells, and 2 µmol/l GAL decreased the number of S phase cells. Both 1 µmol/l and 2 µmol/l GAL increased the number of G0/G1 phase cells. In addition, in a cell apoptosis assay, only 2 µmol/l GAL induced SiHa cell apoptosis. The rates of apoptosis were 8.73% at 24 h and 13.19% at 48 h (Figure 3).

Cell cycle arrest and apoptosis-associated proteins detected by Western blotting. After treatment with GAL (0.5, 1 and 2 µmol/l) for 24 h or 48 h, the expression levels of P53 and P21 were markedly higher than those observed with control treatment (P<0.05). The expression level of CDK-2 was not markedly different between the GAL and control groups. These results suggested that GAL blocked SiHa cells from entering into the S phase. We also found that the expression levels of Bcl-2 in the GAL groups were markedly decreased relative to those in the control group (P<0.05); however, the expression levels of BAX in the GAL groups were obviously elevated relative to those in the control group (P<0.05). The ratios of Bcl-2/BAX in the GAL groups were obviously decreased relative to those in the control group (P<0.05) (Figure 4A-E). In addition, in a caspase-3 activity assay, GAL (0.5, 1 and 2 µmol/l) had no effect on caspase-3 activity in SiHa cells at 24 or 48 hours (Figure 4F).

Inhibition of SiHa cervical cancer xenograft growth in BALB/c nude mice. SiHa cells were inoculated into the left armpit of BALB/c nude mice. After 9 days, mice were intraperitoneally administered 2.5 mg/kg GAL every two days for 10 days. The mice in the control group were administered saline only. Compared with those of the mice in the control group, the body weights of the GAL treated mice were not obviously different (Fig. 5A). However the tumor weight in the GAL treated group was markedly decreased relative to that in the control group (Fig. 5B and C). Treatment with 2.5 mg/kg GAL suppressed the growth of SiHa cervical cancer xenografts by 36.7%. It can be inferred from these results that GAL is a promising candidate for cervical cancer therapy.

Cervical cancer is one of the important causes of cancer-related death in females [14–17]. Evidence from experimental studies and clinical trials has shown that there is great hope for identifying therapeutic cervical cancer drugs from traditional Chinese medicine [18]. Gambogic acid is a Chinese medicine monomer with potent anticancer activity [19]. In this study, we observed that GAL (the lysinate of gambogic acid) inhibited cell proliferation, induced cell apoptosis and increased the number of G0/G1 phase cells in human cervical carcinoma (SiHa cells). Although GAL suppressed the proliferation of both SiHa cells and HeLa cells, GAL had a stronger inhibitory effect on SiHa cells. It was speculated that the possible reason is that the growth of SiHa cells is slower than that of HeLa cells which was confirmed by the growth curves for SiHa and HeLa cells (Figure 2), and GAL has a better inhibitory effect on slowly proliferating cells. In addition, at the higher concentration, GAL induced a small amount of apoptosis in SiHa cells.

The P21 gene, a cyclin-dependent kinase inhibitor, was discovered in recent years [20]. It is not only involved in tumor suppression but can also inhibit the activity of cyclin-dependent kinase (CDK) complexes, thereby regulating the cell cycle, DNA replication and DNA repair [21] and closely linking tumor suppression to the cell cycle control process. P21 and P53 can form a cell cycle G1 checkpoint [22]. After DNA damage, this checkpoint cannot be passed without repair, reducing the replication and accumulation of damaged DNA and thereby exerting a tumor suppressive effect [23]. In this study, we observed that the expression levels of P53 and P21 were markedly increased. We also found that the numbers of SiHa cells in the G0/G1 phase in the GAL groups were increased compared with the number in the control group. Therefore, it could be deduced that GAL can block S-phase entry by G0/G1 phase SiHa cells through activation of the P53-P21 signaling pathway.

When the concentration of GAL was increased, we observed the presence of apoptosis. Apoptosis is a type of cell death essential in the removal of unwanted cells [24, 25]. It is well known that the B-cell lymphoma (Bcl-2) family plays an important role in apoptosis. Members of this family can block pore formation at the mitochondrial outer membrane, thereby inhibiting caspase activation [26, 27]. In the present study we explored the effect of GAL on the Bcl-2/BAX signaling pathway and found that GAL (2 µmol/l) could induce some SiHa cell apoptosis by reducing the expression levels of Bcl-2 and raising the expression levels of BAX. However, 1µmol/l GAL had no effect on cell apoptosis. In previous studies we found that GAL induced breast cancer MCF-7 cell apoptosis through the caspase-3 pathway, while in this study we found that GAL induced cervical cancer SiHa cell apoptosis through the Bcl-2/Bax pathway. The underlying mechanism of this difference may be due to the different tissue sources of these cell lines. In addition, the results of caspase-3 activity assay showed that GAL had no effect on caspase-3 activity in SiHa cells. It was inferred that caspase-3 dose not take part in GAL (2 µmol/l)-induced SiHa cell apoptosis.

In addition to the inhibitory effect of GAL on SiHa cells observed in vitro, we also studied the inhibitory effect of GAL on SiHa tumor growth in vivo. We found that GAL inhibited the growth of SiHa xenograft tumors in nude mice and without significantly effecting mouse weight gain. The results suggest that GAL inhibits tumor growth to a certain extent without in vivo toxicity.

In vivo and in vitro experiments demonstrated that GAL could reduce the proliferation, induce G0/G1 phase arrest and apoptosis of SiHa cells and inhibit tumor growth in mouse xenograft tumors model with low toxicity to the body. GAL is expected to be a promising compound for cervical cancer therapy.

Acknowledgements

We sincerely thank to Mrs. Xiaodong Xu for performing flow cytometric analysis.

Author contributions

Yajun Lin and Yinghua Liu conceptualized the work and supervised the experiments. Jie Niu, Jingyu Sun, and Xin Zhang performed the experiments. Jie Niu, Yajun Lin, Jun Guo, and Yahua Liu contributed significantly to the data analysis and manuscript preparation.

Availability of data and materials

The data and related materials used and/or analyzed in the present study can be obtained from the corresponding author.

Declarations

Ethics approval

The study involving mice was approved by the Institutional Animal Care and Use Committee of Beijing Bozhiyuan Biotechnology Co., Ltd.

Consent for publication

The manuscript was approved for publication by all authors.

Competing financial interests

We declare that there are no competing financial interests.

Funding

This work was supported by grants from the CAMS Innovation Fund for Medical Sciences (No. 2018-I2M-1-002), the National Natural Science Foundation of China (No. 81671391), the Basic Research Project of "health food risk assessment and functional evaluation", the Special Project of "food safety key technology research and development", the National Key Research and Development Plan of the 13^th Five-year Plan (No. 2018YFC1602100), and the Beijing Hospital Nova project (No. BJ-2020-086).

Author details

¹ Peking University Fifth School of Clinical Medicine, Beijing, China

² The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology of National Health Commission, P. R. China

³Emergence Department, the Third Medical Center, Chinese PLA (People's Liberation Army) General Hospital, Beijing, China

⁴Department of Nutrition, the First Medical Center, Chinese PLA General Hospital, Beijing, China.

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Antitumor activity of gambogic acid lysinate in human SiHa cells in vitro and in vivo

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