Hydrogen kills cancer cells in the lungScientific Research

Suppression of autophagy facilitates hydrogen gas‑mediated lung cancer cell apoptosis

Doi:  10.3892/ol.2020.11973

• Leyuan Liu
• Zhenfeng Yan
• Yuanyuan Wang
• Jinghong Meng
• Gang Chen

Abstract

Our previous study found that hydrogen (H2) could effectively inhibit the progression of lung cancer; however, the underlying mechanism has not been elucidated. This study aimed to investigate the role of H2 in autophagy of lung cancer cells, and to reveal the effect of autophagy on H2-mediated apoptosis of lung cancer cells and its underlying mechanism. Expression levels of proteins associated with apoptosis and autophagy were detected using western blot analysis. Autophagy was inhibited by 3-methyladenine treatment or downregulation of Beclin1, while rapamycin was used to induce autophagy. Cell growth and apoptosis were detected using Cell Counting Kit-8 and flow cytometry, respectively. The results showed that apoptosis and autophagy were significantly enhanced in H2-treated A549 and H1975 lung cancer cell lines. However, enhanced autophagy attenuated the apoptosis-promoting effect of H2 and vice versa. Furthermore, it was found that H2 treatment induced a significant decrease in the protein expression levels of phosphorylated STAT3 and Bcl2, and overexpression of STAT3 abolished the role of H2 in promoting apoptosis and autophagy. In conclusion, this study showed that H2 could promote apoptosis and autophagy in lung cancer cells by inhibiting the activation of STAT3/Bcl2 signaling pathway, and inhibiting autophagy could enhance the role of H2 in promoting lung cancer cell apoptosis.

Results

H2 treatment induces a marked increase in apoptosis and autophagy

This study first examined the effects of H2 treatment on apoptosis and autophagy in lung cancer cells. Following H2 treatment in A549 and H1975 cells, cell viability was decreased in a time-dependent manner (Fig. 1A), while apoptotic rates were increased (Fig. 1B and C) compared to controls. The expression levels of pro-apoptotic proteins, including cleaved caspase 3 and cleaved PARP, were significantly increased in both cell lines after H2 treatment in a dose-dependent manner compared to the control group (Fig. 1D and E) (Fig. 1D and E). In addition, Beclin1 expression and LC3BII/I ratio were also significantly increased after H2 treatment, while p62 expression was significantly decreased (Figure 1D and E), also in a dose-dependent manner. These results suggest that H2 can induce apoptosis and autophagy in lung cancer cell lines.

Figure 1. – H2 treatment enhances lung cancer cell apoptosis and autophagy. After A549 and H1975 cells were treated with 20, 40 or 60% H2, the following assays were performed. (A) Cell Counting Kit-8 assay was used to investigate cell growth. (B and C) Flow cytometry assay was used to detect cell apoptosis. (D and E) The protein levels of cleaved-caspase 3, cleaved-PARP, p62, Beclin1, LC3BII and LC3BI were detected using western blot analysis. *P<0.05 and **P<0.01 vs. control. PARP, poly ADP-ribose polymerase; LC3, light chain 3; H2, hydrogen gas.

Autophagy weakens the role of H2 in promoting cell apoptosis in lung cancer cells

Subsequently, the role of autophagy in H2-induced apoptosis of lung cancer cells was examined. When A549 and H1975 cells were treated with the autophagy inducer RAPA, the protein expression levels of Beclin1 and LC3BII/I were significantly increased, while that of p62 was decreased (Figure 2A). Since 60% H2 caused significant changes in cell viability and apoptosis, 60% H2 was chosen for subsequent experiments. After RAPA treatment, the apoptosis rate (Fig. 2B) and the protein expression levels of cleaved caspase 3 and cleaved PARP (Fig. 2C and D) were significantly decreased compared with the H2 group. In contrast, the protein expression levels of Beclin1 and LC3BII/I were significantly decreased and p62 expression was increased after cells were treated with the autophagy inhibitor 3-MA (Figure 2E). Furthermore, 3-MA treatment enhanced H2-mediated increase in apoptosis rates and the protein expression levels of cleaved caspase 3 and cleaved PARP compared to the H2 group (Fig. 2G and H). These results suggest that autophagy attenuates the effect of H2 in inducing apoptosis in lung cancer cells.

Figure 2. – Evaluation of autophagy effects on H2-induced lung cancer cell apoptosis. (A) The protein expression levels of p62, Beclin1, LC3BII and LC3BI, following treatment of A549 and H1975 cells with RAPA were detected using western blot analysis. (B) Cell apoptosis was investigated using a flow cytometry assay. Western blot analysis of the expression of cleaved-caspase 3 and cleaved-PARP in (C) A549 and (D) H1975 cells treated with H2 or H2+RAPA. (E) The protein expression levels of p62, Beclin1, LC3BII and LC3BI were detected using western blot analysis following treatment of A549 and H1975 cells with 3-MA. (F) Cell apoptosis was investigated using flow cytometry. Western blot analysis of the protein expression levels of cleaved-caspase 3 and cleaved-PARP following treatment of (G) A549 and (H) H1975 cells with H2 or H2+3-MA. *P<0.05. **P<0.01. LC3, light chain 3; RAPA, rapamycin; PARP, poly ADP-ribose polymerase; PI, propidium iodide; 3-MA, 3-methyladenine; H2, hydrogen gas.

Knockdown of Beclin1 enhances the roles of H2 in promoting cell apoptosis in lung cancer cell lines

To further elucidate the role of autophagy in H2-mediated apoptosis in lung cancer cells, knockdown experiments were performed. Beclin1 expression was significantly reduced in A549 and H1975 cells transfected with siRNA targeting the human Beclin1 gene. Since si-Beclin1-3 significantly reduced the protein expression level of Beclin1 in the 3 siRNAs (Figure 3A), it was selected for subsequent experiments. After cells were transfected with si-Beclin1-3, the protein expression level of p62 was increased and the expression ratio of LC3BII:LC3BI was decreased (Figure 3B and C). Notably, H2 treatment-induced apoptosis was significantly increased when Beclin1 was silenced in A549 and H1975 cells compared with the H2 group (Fig. 3D).

Furthermore, knockdown of Beclin1 increased the protein expression levels of cleaved caspase 3 and cleaved PARP in A549 and H1975 cells after H2 treatment compared to cells treated with H2 alone (Figure 3E and F) . These results further confirmed that inhibition of autophagy could enhance the effect of H2 on apoptosis induction in lung cancer cell lines.

Figure 3. – Knockdown of Beclin1 enhances the role of H2 in promoting lung cancer cell apoptosis. (A) The knockdown efficiency of si-Beclin1 in A549 and H1975 cells was detected using western blot analysis. (B) Western blot analysis was used to detect the protein expression levels of p62, LC3BII and LC3BI in A549 and H1975 cells and the results were subsequently (C) quantified. (D) Flow cytometry assay was used to investigate cell apoptosis. The protein expression levels of cleaved-caspase 3 and cleaved-PARP were determined using western blot analysis in (E) A549 and (F) H1975 cells. *P<0.05 and **P<0.01. si, short interfering; LC3, light chain 3; PARP, poly ADP-ribose polymerase; PI, propidium iodide; H2, hydrogen gas.

H2 treatment induces lung cancer cell apoptosis and autophagy by repressing the STAT3/Bcl2 signaling pathway

Next, the role of the STAT3/Bcl2 signaling pathway in H2-mediated apoptosis and autophagy in lung cancer cell lines was investigated. In H1975 and A549 cells, the protein expression levels of p-STAT3 and Bcl2 were significantly decreased in a dose-dependent manner after H2 treatment (Figures 4A and B), while STAT3 overexpression abolished this effect (Figures 4C-E). Furthermore, overexpression of STAT3 in H2-treated cells inhibited lung cancer cell apoptosis and neutralized the H2-mediated increase in apoptosis (Figure 4F and G), as well as cleaved caspase 3, cleaved PARP Increased expression of , Beclin1, LC3BII/I, and p62 (all with the H2 group; Figure 4H and I). The above results indicated that H2 treatment promoted apoptosis and autophagy in lung cancer cells by inhibiting the activation of STAT3/Bcl2 pathway.

Figure 4. – H2 treatment increases lung cancer cell apoptosis and autophagy by repressing the activation of the STAT3/Bcl2 signaling pathway. The protein expression levels of STAT3, p-STAT3 and Bcl2 were detected using western blot analysis in (A) A549 and (B) H1975 cells following treatment with different concentrations of H2. (C) STAT3 expression was detected using western blot analysis following transfection with OE-STAT3 or OE-NC. The protein expression levels of STAT3, p-STAT3 and Bcl2 were detected using western blot analysis in (D) A549 and (E) H1975 cells following treatment with H2 and/or transfected with OE-STAT3. Cell apoptosis was investigated using (F) flow cytometry and the results were subsequently (G) quantified. The protein expression levels of cleaved-caspase 3, cleaved-PARP, Beclin1, p62, LC3BII and LC3BI in (H) A549 and (I) H1975 cells were detected using western blot analysis following transfection with OE-STAT3 and/or treated with H2. *P<0.05 and **P<0.01. STAT, signal transducer and activator of transcription; p-, phosphorylated; PARP, poly ADP-ribose polymerase; LC3, light chain 3; H2, hydrogen gas; OE, overexpression; NC, negative control

References

1. Zhao P, Jin Z, Chen Q, Yang T, Chen D, Meng J, Lu X, Gu Z, He Q. Локално генериране на водород за засилена фототермична терапия. Nat Commun. 2018 г .; 9 : 4241. doi: 10.1038/s41467-018-06630-2.
2. Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, Katsura K, Katayama Y, Asoh S, Ohta S. Водородът действа като терапевтичен антиоксидант чрез селективно намаляване на цитотоксичните кислородни радикали. Nat Med. 2007 г .; 13 : 688–694. doi: 10.1038/nm1577.
3. Ono H, Nishijima Y, Adachi N, Sakamoto M, Kudo Y, Nakazawa J, Kaneko K, Nakao A. Лечение с водород (H2) за остри ериматозни кожни заболявания. Доклад на 4 пациенти с данни за безопасност и неконтролирано проучване за осъществимост с измерване на концентрацията на Н2 върху двама доброволци. Med Gas Res. 2012 г .; 2 : 14. doi: 10.1186/2045-9912-2-14.
4. Chen J, Kong X, Mu F, Lu T, Du D, Xu K. Водород-кислородната терапия може да облекчи индуцираната от лъчетерапия загуба на слуха при пациенти с рак на назофаринкса. Ann Palliat Med. 2019; 8 : 746–751. doi: 10.21037/apm.2019.11.18.
5. Dole M, Wilson FR, Fife WP. Хипербарична водородна терапия: Възможно лечение за рак. Наука. 1975; 190 : 152–154. doi: 10.1126/наука.1166304.
6. Wang D, Wang L, Zhang Y, Zhao Y, Chen G. Водородният газ инхибира прогресията на рака на белия дроб чрез насочване към SMC3. Biomed Pharmacother. 2018 г .; 104 : 788-797. doi: 10.1016/j.biopha.2018.05.055.
7. Горди С, Той YW. Кръстосаните връзки между автофагия и апоптоза: докъде води това? Протеинови клетки. 2012 г .; 3 : 17–27. doi: 10.1007/s13238-011-1127-x.
8. Levine B, Kroemer G. Автофагия в патогенезата на болестта. Клетка. 2008 г .; 132 : 27–42. doi: 10.1016/j.cell.2007.12.018.
9. Sotthibundhu A, McDonagh K, von Kriegsheim A, Garcia-Munoz A, Klawiter A, Thompson K, Chauhan KD, Krawczyk J, McInerney V, Dockery P, et al. Рапамицин регулира автофагията и клетъчната адхезия в индуцирани плурипотентни стволови клетки. Стволови клетки Res Ther. 2016 г .; 7 : 166. doi: 10.1186/s13287-016-0425-x.
10. Boya P, Reggiori F, Codogno P. Възникваща регулация и функции на автофагията. Nat Cell Biol. 2013; 15 : 713–720. doi: 10.1038/ncb2815.
11. Levine B. Клетъчна биология: Автофагия и рак. Природата. 2007 г .; 446 : 745–747. doi: 10.1038/446745a.
12. Galluzzi L, Pietrocola F, Bravo-San Pedro JM, Amaravadi RK, Baehrecke EH, Cecconi F, Codogno P, Debnath J, Gewirtz DA, Karantza V, et al. Автофагия при злокачествена трансформация и прогресия на рака. EMBO J. 2015; 34 : 856–880. doi: 10.15252/embj.201490784.
13. Wasik AM, Grabarek J, Pantovic A, Cieślar-Pobuda A, Asgari HR, Bundgaard-Nielsen C, Rafat M, Dixon IM, Gamivi S, Łos MJ. Препрограмиране и канцерогенеза-паралели и различия. Int Rev Cell Mol Biol. 2014 г .; 308 : 167–203. doi: 10.1016/B978-0-12-800097-7.00005-1.
14. Sridhar S, Botbol Y, Macian F, Cuervo AM. Автофагия и болест: Винаги две страни на проблема. Дж. Патол. 2012 г .; 226 : 255–273. doi: 10.1002/пътека.3025.
15. Levy DE, Darnell JE., Jr Stats: Транскрипционен контрол и биологично въздействие. Nat Rev Mol Cell Biol. 2002; 3 : 651–662. doi: 10.1038/nrm909.
16. Akira S, Nishio Y, Inoue M, Wang XJ, Wei S, Matsusaka T, Yoshida K, Sudo T, Naruto M, Kishimoto T. Молекулно клониране на APRF, нов IFN-стимулиран ген фактор 3 p91-свързан транскрипционен фактор участва в gp130-медиирания сигнален път. Клетка. 1994; 77 : 63–71. doi: 10.1016/0092-8674 (94) 90235-6.
17. Niu G, Wright KL, Huang M, Song L, Haura E, Turkson J, Zhang S, Wang T, Sinibaldi D, Coppola D, et al. Конститутивната активност на Stat3 регулира експресията на VEGF и туморната ангиогенеза. Онкоген. 2002; 21 : 2000-2008. doi: 10.1038/sj.onc.1205260.
18. Tong M, Wang J, Jiang N, Pan H, Li D. Връзка между свръхекспресията на p-STAT3 и прогнозата при рак на белия дроб: Систематичен преглед и мета-анализ. PLoS One. 2017 г .; 12 : e0182282. doi: 10.1371/journal.pone.0182282.
19. You L, Wang Z, Li H, Shou J, Jing Z, Xie J, Sui X, Pan H, Han W. Ролята на STAT3 в автофагията. Автофагия. 2015 г .; 11 : 729–739. doi: 10.1080/15548627.2015.10.1017192.
20. Guo S, Luo W, Liu L, Pang X, Zhu H, Liu A, Lu J, Ma DL, Leung CH, Wang Y, Chen X. Изокриптотаншинон, STAT3 инхибитор, индуцира апоптоза и автофагия пред смъртта в A549 белодробни ракови клетки. J Цел за наркотици. 2016 г .; 24 : 934–942. doi: 10.3109/1061186X.2016.1157882.
21. Li S, Liao R, Sheng X, Luo X, Zhang X, Wen X, Zhou J, Peng K. Водороден газ при лечение на рак. Преден Онкол. 2019; 9 : 696. doi: 10.3389/fonc.2019.00696.
22. Li FY, Zhu SX, Wang ZP, Wang H, Zhao Y, Chen GP. Консумацията на вода, богата на водород, предпазва от индуцирана от железен нитрилотриацетат нефротоксичност и ранни промоционни туморни събития при плъхове. Food Chem Toxicol. 2013; 61 : 248–254. doi: 10.1016/j.fct.2013.10.004.
23. Zhou P, Lin B, Wang P, Pan T, Wang S, Chen W, Cheng S, Liu S. Лечебният ефект на богата на водород вода върху остри радиационно-индуцирани кожни наранявания при плъхове. J Radiat Res. 2019; 60 : 17–22. doi: 10.1093/jrr/rry074.
24. Wu Y, Yuan M, Song J, Chen X, Yang H. Водороден газ от лечение на възпаление до терапия на рак. ACS Nano. 2019; 13 : 8505–8511. doi: 10.1021/acsnano.9b05124.
25. Chen JB, Pan ZB, Du DM, Qian W, Ma YY, Mu F, Xu KC. Газотерапията с водород предизвиква свиване на метастатичен рак на жлъчния мехур: Доклад за случая. Случаи на World J Clin. 2019; 7 : 2065–2074. doi: 10.12998/wjcc.v7.i15.2065.
26. Liu G, Pei F, Yang F, Li L, Amin AD, Liu S, Buchan JR, Cho WC. Роля на автофагия и апоптоза при недребноклетъчен рак на белия дроб. Int J Mol Sci. 2017 г .; 18 : 367. doi: 10.3390/ijms18020367.
27. Kroemer G, Mariño G, Levine B. Автофагия и интегрираната реакция на стреса. Mol Cell. 2010 г .; 40 : 280–293. doi: 10.1016/j.molcel.2010.09.023.
28. Dang S, Yu ZM, Zhang CY, Zheng J, Li KL, Wu Y, Qian LL, Yang ZY, Li XR, Zhang Y, Wang RX. Автофагията насърчава апоптоза на мезенхимни стволови клетки при възпалителна микросреда. Стволови клетки Res Ther. 2015 г .; 6 : 247. doi: 10.1186/s13287-015-0245-4.
29. Zhang M, Su L, Xiao Z, Liu X, Liu X. Метилжасмонатът индуцира апоптоза и проапоптотична автофагия чрез ROS пътя при човешки недребноклетъчен рак на белия дроб. Am J Cancer Res. 2016 г .; 6 : 187–199.
30. Galluzzi L, Bravo-San Pedro JM, Vitale I, Aaronson SA, Abrams JM, Adam D, Alnemri ES, Altucci L, Andrews D, Annicchiarico-Petruzzelli M, et al. Съществени и допълнителни аспекти на клетъчната смърт: Препоръки на NCCD 2015 Разликата в клетъчната смърт. 2015 г .; 22 : 58–73. doi: 10.1038/cdd.2014.137.
31. Уайт Е. Ролята на автофагията при рак. J Clin Invest. 2015 г .; 125 : 42–46. doi: 10.1172/JCI73941.
32. Liang L, Hui K, Hu C, Wen Y, Yang S, Zhu P, Wang L, Xia Y, Qiao Y, Sun W, et al. Инхибирането на автофагията потенцира антиангиогенното свойство на мултикиназния инхибитор анлотиниб чрез JAK2/STAT3/VEGFA сигнализиране в недребноклетъчни ракови клетки на белия дроб. J Exp Clin Cancer Res. 2019; 38 : 71. doi: 10.1186/s13046-019-1093-3.
33. Ин C, Zhang H, Liu X, Zhang H, Zhang Y, Bai X, Wang L, Li H, Li X, Zhang S, et al. Намаленият MCOLN1 намалява прогресията на недребноклетъчен рак на белия дроб чрез инхибиране на лизозом-автофагия. Cancer Manag Res. 2019; 11 : 8607–8617. doi: 10.2147/CMAR.S216538.
34. Li Z, Wang Y, Wu L, Dong Y, Zhang J, Chen F, Xie W, Huang J, Lu N. Апуриновата ендонуклеаза 1 насърчава цисплатиновата резистентност на белодробните ракови клетки чрез индуциране на Parkin-медиирана митофагия. Oncol Rep. 2019; 42 : 2245–2254.
35. Fan J, Ren D, Wang J, Liu X, Zhang H, Wu M, Yang G. Bruceine D индуцира апоптоза и автофагия на рак на белия дроб чрез сигналния път ROS/MAPK in vitro и in vivo. Dis клетъчна смърт Dis. 2020 г .; 11 : 126. doi: 10.1038/s41419-020-2317-3.
36. El-Khattouti A, Selimovic D, Haikel Y, Hassan M. Crosstalk между апоптоза и автофагия: Молекулярни механизми и терапевтични стратегии при рак. J клетъчна смърт. 2013; 6 : 37–55. doi: 10.4137/JCD.S11034.
37. Торбърн А. Апоптоза и автофагия: Регулаторни връзки между два предполагаемо различни процеса. Апоптоза. 2008 г .; 13 : 1–9. doi: 10.1007/s10495-007-0154-9.
38. Гупта Н. А., Колачала В. Л., Дзян Р, Абрамовски С, Шеной А, Костърс А, Павулури Н, Анания Ф, Кърк АД. Намаляването на автофагията подобрява хепатоцелуларното увреждане след исхемично-реперфузионно увреждане в миши стеатотичен черен дроб. Am J Physiol Gastrointest Черен дроб Physiol. 2014 г .; 307 : G1088 – G1099. doi: 10.1152/ajpgi.00210.2014.
39. Guan P, Sun ZM, Luo LF, Zhou J, Yang S, Zhao YS, Yu FY, An JR, Wang N, Ji ES. Водородът предпазва от хронична интермитентна хипоксия, причинена от бъбречна дисфункция, като насърчава автофагията и облекчава апоптозата. Life Sci. 2019; 225 : 46–54. doi: 10.1016/j.lfs.2019.04.005.
40. Yan M, Yu Y, Mao X, Feng J, Wang Y, Chen H, Xie K, Yu Y. Вдишването на водороден газ отслабва индуцираното от сепсис увреждане на черния дроб по FUNDC1-зависим начин. Int Immunopharmacol. 2019; 71 : 61–67. doi: 10.1016/j.intimp.2019.03.021.
41. Gao Y, Yang H, Chi J, Xu Q, Zhao L, Yang W, Liu W, Yang W. Водородният газ намалява реперфузионното увреждане на миокардната исхемия, независимо от посткондиционирането при плъхове, като намалява автофагията, предизвикана от ендоплазмен ретикулум. Клетъчен биохимичен физиол. 2017 г .; 43 : 1503–1514. doi: 10.1159/000481974.
42. Liu Y, Gong W, Yang ZY, Zhou XS, Gong C, Zhang TR, Wei X, Ma D, Ye F, Gao QL. Кверцетин индуцира защитна автофагия и апоптоза чрез ER стрес чрез оста p-STAT3/Bcl-2 при рак на яйчниците. Апоптоза. 2017 г .; 22 : 544–557. doi: 10.1007/s10495-016-1334-2.
43. Liu K, Ren T, Huang Y, Sun K, Bao X, Wang S, Zheng B, Guo W. Апатиниб насърчава автофагия и апоптоза чрез VEGFR2/STAT3/BCL-2 сигнализиране при остеосаркома. Dis клетъчна смърт Dis. 2017 г .; 8 : e3015. doi: 10.1038/cddis.2017.422.
44. Bai X, Liu S, Yuan L, Xie Y, Li T, Wang L, Wang X, Zhang T, Qin S, Song G, et al. Богатият на водород физиологичен разтвор медиира неврозащитата чрез регулиране на стреса на ендоплазмения ретикулум и автофагията при хипоксия-исхемия при новородено мозъчно увреждане при мишки. Brain Res. 2016 г .; 1646 : 410–417. doi: 10.1016/j.brainres.2016.06.020.