Inhibition of urethane-induced lung carcinogenesis in mice by a Rhizoma paridis saponin involved EGFR/PI3K/Akt pathway

Abstract

Lung cancer is one of the most common cancers in the world. Due to a lack of successful treatments for lung cancer, there is a need to evaluate new and effective agents for lung cancer treatment. Rhizoma Paridis saponins (RPS) were deemed effective for the treatment of cancer disease. In the present study, we evaluated the role of RPS on urethane-induced lung carcinogenesis in C57BL/6 mice. As a result, the treatment with RPS reduced the severity of the histopathology on urethane-induced lung tumorigenesis in mice. The mechanisms of its antitumor effect involved in (i) reducing oxidative stress injury through up-regulation of Nrf2 and HO-1, (ii) decreasing the level of inflammatory factors, like COX-2 and PGE2, (iii) activation of caspase-3 through the down-regulation of anti-apoptotic protein (Bcl2) and up-regulation of pro-apoptotic protein (Bax), (iv) decreasing the expression of PCNA, and (v) inhibiting the epidermal growth factor receptor (EGFR)/PI3K/Akt pathway. In conclusion, RPS would be a potent agent inhibiting lung cancer in the future.

---

1. Introduction

Lung cancer is the most common diagnosed malignancies and the leading cause of cancer death in China.¹ It's reported to be associated with risk factors including cigarette smoking, body mass index, environmental carcinogen exposure and so forth.²

Urethane, also called ethyl carbamate (EC), is regarded as one of the chemical carcinogens in cigarettes³ and a genotoxic carcinogen in fermented foods and beverages.⁴ It's oxidized to vinyl carbamate (VC), and further transformed to an epoxide through mediation of CYP2E1.⁵ The metabolism of EC and VC initiates the cascade of events leading to pulmonary adenomas formation in humans and mice.⁶ Moreover, pulmonary adenoma formation in this model also results from abrogation of mitochondria,⁷ lasting glycolytic stress of lung and oxidative stress,⁸ a series of genetic and epigenetic alterations in pulmonary epithelial cells, especial for epidermal growth factor receptor (EGFR) overexpression⁹ and so on. Hence, EC-induced lung carcinogenesis is considered as a valuable model to study antitumor effects of various natural or synthetic compounds.¹⁰

Paris polyphylla var. yunnanensis (Fr.) Hand.-Mazz. (PPY), is a traditional Chinese medicinal herb that has been used in treating cancer for many years.¹¹ As previously reported, Rhizoma paridis Saponins (RPS), as its effective part, showed strong anti-lung cancer effects through enhancement of immunostimulation and induction of apoptosis,¹² and antimetastatic activities by up-regulation of TIMP-2 and down-regulation of MMP-2 and MMP-9 levels¹³ in Lewis tumor-bearing C57BL/6 mice. In addition, RPS inhibited diethylnitrosamine (DEN) induced pulmonary adenoma and attenuated hepatotoxicity.¹⁴ All these indicated RPS would be a potent anti-lung cancer agent used in the prospective application. In this experiments, we first used urethane-induced lung carcinogenesis models to examine the underlying mechanism of antitumor effects of RPS.

2. Materials and methods

2.1 Reagents

The dried rhizoma of Paris polyphylla var. yunnanensis was collected in September 2013 from Lijiang, Yunnan Province, China, and identified by Professor Gao Wenyuan. A voucher specimen (GWCL201309) was deposited at the School of Pharmaceutical Science and Technology at Tianjin University, Tianjin, China. RPS was prepared as previously described.¹⁵

2.2 Animal maintenance and treatments

Mice experiments were conducted under ethics approval from Institutional Animal Care and Use Committee of Institute of Radiation Medicine Chinese Academy of Medical Sciences (Permit Number: IACUC2014-010). Female C57BL/6 mice, with body weight 18.0 ± 2.0 g, were obtained from Beijing Experimental Animal Center. This study was carried out in accordance with the Regulation for the Administration of Affairs Concerning Experimental Animals. Twenty-four mice were randomly assigned into three groups as follows (Fig. 1). Group I: injected with saline solution and oral saline solution, used as a negative control group (normal group). Group II: injected with urethane (800 mg per kg body weight per mouse, twice a week for five weeks) and oral saline solution, used as a positive control group (model group). Group III: injected with urethane (800 mg per kg body weight per mouse, twice a week for five weeks) and oral RPS (50 mg per 10 mL per kg body weight per mouse, every other day starting at the 1^st week of urethane injection) (RPS group).

Fig. 1 Experimental design to assess the inhibition rate of urethane-induced lung carcinogenesis in C57BL/6 mice by RPS.

Blood samples (0.5 mL) were collected into heparinized tubes from each mouse by the puncture of the retro-orbital sinus in the 18^th week. Blood was immediately processed for plasma by centrifugation at 3500g for 15 minutes. Plasma was frozen and maintained at −20 °C until analyses.

2.3 Histopathological examination

For the histopathological examination, portions of lung tissues were fixed in 10% formalin. After proper dehydration, the lung tissues were embedded in paraffin wax. Five μm-thick sections were prepared and stained with hematoxylin and eosin. Every organ was randomly cut into 5 histological sections. Histopathology examination was completed using Nikon eclipse TE2000-U Microscope and performed by a pathologist who was unaware of whether tissues were treated.

2.4 Measurements of 8-OHdG, COX-2, and PGE2 levels by ELISA assays

Plasma samples were analyzed for 8-hydroxy-deoxyguanosine (8-OHdG) (Huiying Co., Shanghai, China), cyclooxygenase-2 (COX-2) and particularly prostaglandin E2 (PGE2) (Huole Co., Shanghai, China) with mouse ELISA kits by following the manufactures' instructions. Each sample was assayed in duplicate. The values were within the linear portion of the standard curve.

2.5 Western blot assay

Total proteins from lung tissues were isolated using the tissue protein extraction kit (Bio-Rad, USA). The obtained protein was quantified by the Bradford Assay Kit (BioRad, Hercules, CA). Then protein samples (20–100 μg) were separated on 12% SDS-PAGE and transferred to PVDF membranes (Millipore Corp., USA), with being probed with β-actin, p-AKT, PCNA (Bioworld), p-mTOR (Cell Signaling), Nrf2, HO-1, EGFR, p-EGFR, p-PI3K, Caspase 3, Bax (Santa Cruz Biotechnology) and Bcl-2 (Boster) antibodies followed by appropriate secondary antibody. Equal protein loading was checked by quantifying β-actin. The relative optical densities of the bands were quantified using Odyssey infrared imaging system (LI-COR Biotechnology, USA).

2.6 Statistical analysis

Statistical evaluation was conducted by using SPSS 17.0 for Windows package software. Data have been expressed as the means ± standard error means (SEM). One-way variance analysis and Duncan multiple range test were used to determine significantly different groups. P values less than 0.05 were considered as significant differences for all statistical calculations.

3. Results

3.1 General observations and histopathological examination

As previous reported, consequent injection of urethane played an important role in the development of pulmonary adenoma in C57BL/6 mice.¹⁰ There was a decrease for the body weight and body temperature in urethane-injected mice. Pathological examination proved that no abnormal findings in the lungs of normal mice (Fig. 2A). However, there were infiltrations of inflammatory cells, alveolar epithelial hyperplasia and irregular cellular nodules in urethane-induced lungs. Some monomorphic cuboidal cells forming tubulopapillary structures in the lung were regarded as pulmonary adenoma (Fig. 2B). RPS significantly decreased the number and the area of the tumor embolus in lung tissues and relieved infiltration of inflammatory cells and alveolar septal thickening (Fig. 2C).

Fig. 2 Histopathology of lung tissues. Paraffin-embedded sections were stained with H&E. Representative photomicrographs showed (A) normal lung tissue, (B) model lung tissue and (C) RPS-treated lung tissue. Arrows indicated representative images of adenoma in tissues. Higher magnification of area indicated by circles. Upper panels, magnification: 400×. Lower panels, magnification: 100×.

3.2 CYP2E1 expression and oxidative stress affected by RPS

As previous reports, CYP2E1-mediated oxidation played an essential role in urethane-induced carcinogenicity.¹⁶ Meanwhile, 8-OHdG as a marker of oxidative DNA damage, both of them were significantly elevated in urethane-treated mice (p < 0.05). However, treatment with RPS decreased their levels (Fig. 3A and B). To further investigate the molecular mechanism of anti-oxidant stress, we measured the expression levels of Nrf2 and HO-1. HO-1 as an important detoxification enzyme displayed anti-oxidant, detoxification, anti-inflammatory and cytoprotective activity. Nrf2 as a vital nuclear factor activated the genes of antioxidant proteins and detoxifying enzymes. Both of them were decreased in model group, while up-regulated in RPS-treated ones.

Fig. 3 Effect of RPS on urethane-induced pulmonary adenocarcinoma in mice. (A) Expressive comparison of CYP2E1 in liver tissues. (B) The serum level of 8-OHdG and protein expression of HO-1 and Nrf2 in lung tissues among three groups. Different letters meant significant differences between two groups. Data were shown as means ± SEM. (C) Effects of RPS on the levels of COX2 and PGE2 in the serum (C1) and lung tissues (C2) of three groups. Different letters meant significant differences between two groups. Data were shown as means ± SEM. (D) Effect of RPS on the protein expression of cell proliferation and apoptosis related proteins in lung tissues. Different letters meant significant differences between two groups. Data were shown as means ± SEM of three independent experiments. (E) Western blot analyzed the expression of EGFR, PI3K/Akt/mTOR and MAPK (p-P38, p-JNK and p-Erk1/2) among the three groups. (F) Proposed pathway of RPS inhibiting urethane-induced pulmonary adenomas formation in mice.

3.3 Inflammatory factors

Urethane increased the expression of COX-2 and PGE2 compared with the normal group both in serum and lung tissues. Meanwhile, RPS significantly decreased both of their levels compared with non-treated urethane-injected mice except the level of COX-2 in lung tissue (Fig. 3C).

3.4 Apoptotic pathway in lung tissues

As Fig. 3D indicated, urethane induced pulmonary adenomas formation in mice through the up-regulating protein levels of PCNA and anti-apoptotic protein Bcl-2 (p < 0.05). Meanwhile, RPS significantly reduced urethane induced PCNA and Bcl-2 expression in lung tissues and remarkably increased levels of cleaved caspase-3 and Bax, which were associated with the apoptosis pathway.

3.5 Effect of RPS on urethane induced lung tumors associated with EGFR pathway

It's reported that pulmonary adenomas formation in urethane-induced model results from a series of genetic and epigenetic alterations in pulmonary epithelial cells, especial for EGFR over expression.⁹ Therefore, in this experiment, we focused on EGFR pathway on the protein levels. As a result, RPS blocked urethane-induced EGFR over expression, which was associated with the inhibition of the PI3K/Akt/mTOR pathway (Fig. 3E). However, there seemed no effects on the MAPK proteins, such as JNK, p38 and Erk1/2.

4. Discussion

Nearly all lung cancers are associated with exposure to environmental carcinogens. Urethane as one of the most likely candidates for causing additional carcinogenicity has been judged as a probable health risk for regular populations of fermented foods and beverages.⁴ Naturally-derived and inspired products (small molecules, proteins and nucleic acids) have been recognized to be of high importance in sensing, diagnosis and treatment.^17,18 The development of cancer treatments has greatly benefited from natural products. It has been reported that almost half of small molecule anti-cancer drugs approved by U.S. FDA since 1940s were actually natural products related.^19,20

In this research, with the consequent injection of urethane in C57BL/6 mice, there was a decrease in the body weight and body temperature, and the development of pulmonary adenoma (Fig. 2). However, RPS significantly decreased the number and the area of the tumor embolus in lung tissues and relieved infiltration of inflammatory cells and alveolar septal thickening (Fig. 2C).

As previous report, CYP2E1-mediated oxidation played an essential role in urethane-induced carcinogenicity.^6,16 RPS efficiently reduced the level of Cyp2e1 protein to reduce the metabolism of urethane (Fig. 3A). Meanwhile, pulmonary adenomas formation in this model also resulted from oxidant stress.⁸ 8-OHdG as a marker of oxidative DNA damage⁸ was significantly elevated in urethane-treated mice. Nrf2 prevented malignant progression through mitigating insults from both exogenous and endogenous sources.²¹ HO-1 as one of its target proteins maintained the cellular redox capacity and eliminated reactive oxygen species.²¹ Therefore, Nrf2 activators benefited lung cancer prevention.²² RPS inhibited the formation of 8-OHdG and increased in protein levels of Nrf2 and HO-1 (Fig. 3B). In addition, COX-2 directed the synthesis of prostaglandins including PGE-2 which was linked to the inflammation²³ and potentiated pulmonary adenocarcinogenesis in urethane-induced mice.²⁴ Our results indicated that RPS exhibited an anti-inflammative effect by decreasing of COX2 and PGE2 production (Fig. 3C). Hence, these results proved that RPS, at least in part, participated in the protection against DNA damage and pulmonary adenoma progression via the inhibition of oxidative stress and inflammation formation.

Pulmonary adenomas formation in this model also results from a series of genetic and epigenetic alterations in pulmonary epithelial cells, especial for the over expression of EGFR,⁹ PCNA and anti-apoptotic proteins like Bcl-2 (ref. 25) (Fig. 3F). RPS reduced this trend. Up-regulated pro-apoptotic protein Bax and activated caspase-3 to suppress the rapid pulmonary adenomas cell proliferation (Fig. 3D), which might be through lessening the downstream pathway of EGFR,²⁶ PI3K/Akt/mTOR (Fig. 3E). However, there seemed no remarkable influence on the expression of ERK1/2, p38 and JNK. PI3K/Akt/mTOR pathway could promote cell proliferation, survival, differentiation and so on.²⁷ Thus, RPS could be a promising chemopreventive agent for lung tumorigenesis mainly through the suppression of EGFR/PI3K/AKT pathway.

Overall, RPS inhibited pulmonary adenocarcinoma formation through the protection against oxidative stress injury and inflammatory factors product in the urethane-induced mice. The anti-tumor effect of RPS was also attributed to the suppression of PI3K/Akt/mTOR and down-regulation of EGFR to activate apoptosis in lung tumor cells. In conclusion, our work enriched the therapeutic utility of saponins and indicated that RPS would be a potent agent inhibiting lung cancer in the future.

Conflict of interest

We declared that there were no conflicts of interest in this paper.

Abbreviations

8-OHdG	8-Hydroxy-deoxyguanosine
COX-2	Cyclooxygenase-2
EGFR	Epidermal growth factor receptor
ELISA	Enzyme-linked immunosorbent assay
HO-1	Heme oxygenase-1
MAPK	Mitogen-activated protein kinase
Nrf-2	Nuclear factor-2 erythroid related factor-2
PCNA	Proliferating cell nuclear antigen
PGE2	Particularly prostaglandin E2
PI3K	Phosphatidylinositol-3-kinase
RPS	Rhizoma paridis saponins

Acknowledgements

This work was supported by grants 81673647, 81503086 and 81373904 from National Natural Science Foundation of China and Drug Creation Project 2014ZX09301307-018 from Science and Technology in China.

References

1. W. Chen, R. Zheng, P. D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X. Q. Yu and J. He, Ca-Cancer J. Clin., 2016, 66, 115–132.
2. T. Kawaguchi, Y. Koh, M. Ando, N. Ito, S. Takeo, H. Adachi, T. Tagawa, S. Kakegawa, M. Yamashita, K. Kataoka, Y. Ichinose, Y. Takeuchi, M. Serizawa, A. Tamiya, S. Shimizu, N. Yoshimoto, A. Kubo, S. Isa, H. Saka and A. Matsumura, J. Clin. Oncol., 2016, 34, 2247–2257.
3. L. C. Pulling, B. R. Vuillemenot, J. A. Hutt, T. R. Devereux and S. A. Belinsky, Cancer Res., 2004, 64, 3844–3848.
4. D. W. Lachenmeier, Anal. Bioanal. Chem., 2005, 382, 1407–1412.
5. F. A. Beland, R. W. Benson, P. W. Mellick, R. M. Kovatch, D. W. Roberts, J. L. Fang and D. R. Doerge, Food Chem. Toxicol., 2005, 43, 1–19.
6. P. G. Forkert, Drug Metab. Rev., 2010, 42, 355–378.
7. G. Du, T. Sun, Y. Zhang, H. Lin, J. Li, W. Liu, Y. Wang, B. Zhao, H. Li and Y. Liu, Eur. J. Pharmacol., 2013, 715, 395–404.
8. X. Ma, J. Deng, N. Cao, Z. Guo, Y. Zheng, S. Geng, M. Meng, H. Lin, Y. Duan and G. Du, Toxicol. Lett., 2016, 240, 130–139.
9. R. J. Chen, S. J. Tsai, C. T. Ho, M. H. Pan, Y. S. Ho, C. H. Wu and Y. J. Wang, J. Agric. Food Chem., 2012, 60, 11533–11541.
10. Y. E. Miller, L. D. Dwyer-Nield, R. L. Keith, M. Le, W. A. Franklin and A. M. Malkinson, Cancer Lett., 2003, 198, 139–144.
11. S. Man, Y. Wang, Y. Li, W. Gao, X. Huang and C. Ma, Chin. Herb. Med., 2013, 5, 33–46.
12. Y. F. Peng, Y. H. Shi, Z. B. Ding, J. Zhou, S. J. Qiu, B. Hui, C. Y. Gu, H. Yang, W. R. Liu and J. Fan, PLoS One, 2013, 8, e53072.
13. S. Man, W. Gao, Y. Zhang, L. Yan, C. Ma, C. Liu and L. Huang, Steroids, 2009, 74, 1051–1056.
14. S. Man, J. Li, W. Fan, H. Chai, Z. Liu and W. Gao, J. Steroid Biochem. Mol. Biol., 2015, 154, 62–67.
15. Z. Liu, J. Wang, W. Gao, S. Man, Y. Wang and C. Liu, Pharm. Biol., 2013, 51, 899–905.
16. B. I. Ghanayem, Toxicol. Sci., 2007, 95, 331–339.
17. L. Ma, A. Bartholome, M. H. Tong, Z. W. Qin, Y. Yu, T. Shepherd, K. Kyeremeh, H. Deng and D. O'Hagan, Chem. Sci., 2015, 6, 1414–1419.
18. L. Ma and J. Liu, J. Ethnopharmacol., 2014, 158A, 358–363.
19. L. Ma and A. Diao, Anti-Cancer Agents Med. Chem., 2015, 15, 298–306.
20. D. J. Newman and G. M. Cragg, J. Nat. Prod., 2016, 79, 629–661.
21. T. Shen, T. Jiang, M. Long, J. Chen, D. M. Ren, P. K. Wong, E. Chapman, B. Zhou and D. D. Zhang, Antioxid. Redox Signaling, 2015, 23, 651–664.
22. H. Satoh, T. Moriguchi, J. Takai, M. Ebina and M. Yamamoto, Cancer Res., 2013, 73, 4158–4168.
23. C. Cao, R. Gao, M. Zhang, A. L. Amelio, M. Fallahi, Z. Chen, Y. Gu, C. Hu, E. A. Welsh, B. E. Engel, E. B. Haura, W. D. Cress, L. Wu, M. Zajac-Kaye and F. J. Kaye, J. Natl. Cancer Inst., 2015, 107, 358.
24. G. T. Stathopoulos, T. P. Sherrill, D. S. Cheng, R. M. Scoggins, W. Han, V. V. Polosukhin, L. Connelly, F. E. Yull, B. Fingleton and T. S. Blackwell, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 18514–18519.
25. H. C. Pal, S. Sharma, L. R. Strickland, J. Agarwal, M. Athar, C. A. Elmets and F. Afaq, PLoS One, 2013, 8, e77270.
26. Y. Zhang, L. Wang, M. Zhang, M. Jin, C. Bai and X. Wang, J. Cell. Physiol., 2012, 227, 35–43.
27. J. T. Beck, A. Ismail and C. Tolomeo, Cancer Treat. Rev., 2014, 40, 980–989.

---