Effect and mechanism of rapamycin on proliferation and apoptosis of human lung cancer cells

Wenning Cao; Lan Ma

doi:10.14715/cmb/2020.66.6.12

Issue

Vol. 66 No. 6: Issue 6

Issue Published : October 6, 2020

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Effect and mechanism of rapamycin on proliferation and apoptosis of human lung cancer cells

https://doi.org/10.14715/cmb/2020.66.6.12

Wenning Cao

Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China;

Lan Ma

State Key Laboratory of Chemical Oncogenomics, Division of Life Science and Health, The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P. R. China

Corresponding Author(s) : Wenning Cao

howard8863@yandex.com

Cellular and Molecular Biology, Vol. 66 No. 6: Issue 6
Article Published : September 30, 2020

Abstract

The purpose of this study was to investigate the effects of RAPA on the proliferation and the expression of p53, Bcl-2 and Bax proteins in cultured human small cell lung cancer (NCI-H446) cells, and to explore the possible mechanism of RAPA-treated NCI-H446 cells with different concentrations of RAPA-treated NCI-H446 cells. The proliferation of NCI-H446 cells in all groups was assayed by the CCK-8 method. FITC-Annexin V/PI double staining method was used to determine the apoptosis of NCI-H446 cells. The immunohistochemical SP method was used to detect the expression of p53, Bcl-2 and Bax. Expression of p53, Bcl-2 and Bax mRNA was detected by RT-PCR. The results showed that, after 48h treatment, the proliferation of NCI-H446 cells treated with 5ng/mL, 10ng/mL and 15ng/mL RAPA decreased significantly (P < 0.05) and the proliferation inhibition rate increased significantly (P < 0.05) compared with the control group, and the proliferation inhibition rate had a dose-dependent relationship with RAPA. Compared with the control group, the apoptosis rate of NCI-H446 cells treated with 5ng/mL, 10ng/mL and 15ng/mL RAPA increased significantly (P < 0.05), and there was a dose-dependent relationship between the apoptosis rate and RAPA. The expression of Bcl-2 protein and mRNA was higher in the control group, while the expression of p53 and Bax protein and mRNA was lower. The expression of Bcl-2 protein and mRNA decreased and the expression of p53 and Bax protein and mRNA increased gradually with the increase of concentration and the prolongation of action time in 5ng/mL, 10ng/mL and 15ng/mL RAPA groups. In the control group, the intracellular Ca²⁺ concentration was constant, and there was no significant change with time; while in the 5ng / mL, 10ng / mL, and 15ng / mL RAPA group, the intracellular Ca²⁺ concentration in the RAPA group increased significantly after 12 h of administration (P <0.05); After that, with the prolonged action time of the medicine, the intracellular Ca²⁺ concentration in the 5ng / mL, 10ng / mL, and 15ng / mL RAPA group decreased, but at 72h, the effect was 5ng / mL, 10ng / mL, and 15ng / mL RAPA. The intracellular Ca²⁺ fluorescence intensity in the group was still significantly higher than that in the control group (P <0.05). In conclusion, RAPA can induce apoptosis of NCI-H446 cells by down-regulating Bcl-2 gene expression, up-regulating P53 and Bax gene expression, and increasing intracellular Ca²⁺ concentration and its apoptosis induction effect have timeliness and dose-effect.

Keywords

Lung cancer Rapamycin Cell proliferation Apoptosis Bcl-2 p53 Bax.

Cao, W., & Ma, L. (2020). Effect and mechanism of rapamycin on proliferation and apoptosis of human lung cancer cells. Cellular and Molecular Biology, 66(6), 65–70. https://doi.org/10.14715/cmb/2020.66.6.12

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References

Prezzano KM, Ma SJ, Hermann GM. Stereotactic body radiation therapy for non-small cell lung cancer: A review. World J Clin Oncol 2019; 10(1):17-30.
Lembicz M, Gabryel P, Brajer-Luftmann B, Dyszkiewicz W, Batura-Gabryel H. Comorbidities with non-small cell lung cancer: Is there an interdisciplinary consensus needed to qualify patients for surgical treatment? Ann Thorac Med 2018; 13(2):101.
Di Benedetto F, Di Sandro S, De Ruvo N, Montalti R, Ballarin R, Guerrini GP, Spaggiari M, Guaraldi G, Gerunda G. First report on a series of HIV patients undergoing rapamycin monotherapy after liver transplantation. Transplant 2010; 89(6):733-8.
Turska"Kmieć A, Jankowska I, PawÅ‚owska J, KaliciÅ„ski P, Kawalec W, Tomyn M, Markiewicz M, Teisseyre J, Czubkowski P, Rć™kawek J, Socha J. Reversal of tacrolimus"related hypertrophic cardiomyopathy after conversion to rapamycin in a pediatric liver transplant recipient. Pediatr Transplant 2007; 11(3):319-23.
Alamo JM, Barrera L, Casado MD, Bernal C, Marin LM, Suarez G, Sanchez-Moreno L, Jimenez R, Suarez-Grau JM, Sousa JM, Cordero E. Efficacy, tolerance, and safety of mammalian target of rapamycin inhibitors as rescue immunosuppressants in liver transplantation. Transplant Proc 2009; 41: 6: 2181-2183).
Wang D, Gao L, Liu X, Yuan C, Wang G. Improved antitumor effect of ionizing radiation in combination with rapamycin for treating nasopharyngeal carcinoma. Oncol Lett 2017; 14(1):1105-8.
Liu PY, Zhang WB, Wang J, Shen YH, Wei YY. Inhibitory effect and significance of rapamycin on the mammalian target of rapamycin signaling pathway in osteosarcoma stem cells and osteosarcoma cells. Zhonghua Zhong Liu Za Zhi 2013; 35(3):175-80.
Bressac B, Puisieux A, Kew M, Volkmann M, Bozcall S, Mura JB, de la Monte S, Carlson R, Blum H, Wands J, Takahashi H. p53 mutation in hepatocellular carcinoma after aflatoxin exposure. Lancet 1991; 338(8779):1356-9.
Li L, Deng B, Xing G, Teng Y, Tian C, Cheng X, Yin X, Yang J, Gao X, Zhu Y, Sun Q. PACT is a negative regulator of p53 and essential for cell growth and embryonic development. Proc Natl Acad Sci 2007; 104(19):7951-6.
Kalemkerian GP, Ou X, Adil MR, Rosati R, Khoulani MM, Madan SK, Pettit GR. Activity of dolastatin 10 against small-cell lung cancer in vitro and in vivo: induction of apoptosis and bcl-2 modification. Cancer Chemother Pharmacol 1999; 43(6):507-15.
Miyake H, Hara S, Arakawa S, Kamidono S, Hara I. Overexpression of bcl"2 regulates sodium butyrate"and/or docetaxel"induced apoptosis in human bladder cancer cells both in vitro and in vivo. Int J Cancer 2001; 93(1):26-32.
Xiao ZM, Li LJ, Lu ZN, Li CY, Zheng JQ. Effects of extracellular Ca2+ influx and intracellular Ca2+ release on ethanol-induced cytoplasmic Ca2+ overload in cultured superior cervical ganglion neurons. Neurosci Lett 2005; 390(2):98-103.
Wang XZ, Zhang SJ, Chen YX, Chen ZX, Huang YH, Zhang LJ. Effects of platelet-derived growth factor and interleukin-10 on Fas/Fas-ligand and Bcl-2/Bax mRNA expression in rat hepatic stellate cells in vitro. World J Gastroenterol 2004; 10(18):2706.
Fuchs BC, Finger RE, Onan MC, Bode BP. ASCT2 silencing regulates mammalian target-of-rapamycin growth and survival signaling in human hepatoma cells. Am J Physiol Cell Physiol 2007; 293(1): C55-63.
Pencreach E, Guérin E, Nicolet C, Lelong-Rebel I, Voegeli AC, Oudet P, Larsen AK, Gaub MP, Guenot D. Marked activity of irinotecan and rapamycin combination toward colon cancer cells in vivo and in vitro is mediated through cooperative modulation of the mammalian target of rapamycin/hypoxia-inducible factor-1Î± axis. Clin Cancer Res 2009; 15(4):1297-307.
Jiang RY, Pei HL, Gu WD, Huang J, Wang ZG. Autophagic inhibitor attenuates rapamycin-induced inhibition of proliferation in cultured A549 lung cancer cells. Eur Rev Med Pharmacol Sci 2014; 18(6):806-10.
Webster NJ, Resnik JL, Reichart DB, Strauss B, Haas M, Seely BL. Repression of the insulin receptor promoter by the tumor suppressor gene product p53: a possible mechanism for receptor overexpression in breast cancer. Cancer Res 1996; 56(12):2781-8.
Li J, Jiang K, Zhao F. Icariin regulates the proliferation and apoptosis of human ovarian cancer cells through microRNA-21 by targeting PTEN, RECK and Bcl-2. Oncol Rep 2015; 33(6):2829-36.
Lee HJ, Wang CJ, Kuo HC, Chou FP, Jean LF, Tseng TH. Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of Bax/Bak and activation of JNK. Toxicol Appl Pharmacol 2005; 203(2):124-31.
Park KH, Kim SY, Gul R, Kim BJ, Jang KY, Chung HT, Sohn DH. Fatty acids ameliorate doxorubicin-induced intracellular Ca2+ increase and apoptosis in rat cardiomyocytes. Biol Pharm Bull 2008; 31(5):809-15.
Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, Reed JC. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Sci 1999; 284(5412):339-43.
Li M, Kondo T, Zhao QL, Li FJ, Tanabe K, Arai Y, Zhou ZC, Kasuya M. Apoptosis induced by cadmium in human lymphoma U937 cells through Ca2+-calpain and caspase-mitochondria-dependent pathways. J Biol Chem 2000; 275(50):39702-9.
Akbari F, Arminian A, Kahrizi D, Fazeli A, Ghaheri M. Effect of nitrogen sources on gene expression of Stevia rebaudiana (Bertoni) under in vitro conditions. Cell Mol Biol, 64(2): 11-16.
Akbarabadi A, Ismaili A, Kahrizi D, Firouzabadi FN. Validation of expression stability of reference genes in response to herbicide stress in wild oat (Avena ludoviciana). Cell Mol Biol 2018; 64(4). 113-118.
Ghorbani T, Kahrizi D, Saeidi M, Arji I. Effect of sucrose concentrations on Stevia rebaudiana Bertoni tissue culture and gene expression. Cell Mol Biol, 63(11): 32-36.
Ghaheri M, Adibrad E, Safavi SM, Kahrizi D, Soroush A, Muhammadi S, Ghorbani T, Sabzevari A, Ansarypour Z, Rahmanian E. Effects of life cycle and leaves location on gene expression and glycoside biosynthesis pathway in Stevia rebaudiana Bertoni. Cell Mol Biol, 64(2): 17-22.
Kahrizi D, Ghaheri M, Yari Z, Yari K, Bahraminejad S. Investigation of different concentrations of MS media effects on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni. Cell Mol Biol, 64(2): 23-27.
Hashempoor S, Ghaheri M, Kahrizi D, Kazemi N, Muhammadi S, Safavi SM, Ghorbani T, Rahmanian E, Heshmatpanaah M. Effects of different concentrations of mannitol on gene expression in Stevia rebaudiana Bertoni. Cell Mol Biol, 64(2): 28-31.
Kahrizi D, Ghari SM, Ghaheri M, Fallah F, Ghorbani T, Beheshti AA, Kazemi E, Ansarypour Z. Effect of KH2PO4 on gene expression, morphological and biochemical characteristics of stevia rebaudiana Bertoni under in vitro conditions. Cell Mol Biol, 63(7): 107-111.
Fallah F, Nokhasi F, Ghaheri M, Kahrizi D, Beheshti Ale AA, Ghorbani T, Kazemi E, Ansarypour Z. Effect of salinity on gene expression, morphological and biochemical characteristics of Stevia rebaudiana Bertoni under in vitro conditions. Cell Mol Biol 2017; 63(7): 102-106.
Bordbar M, Darvishzadeh R, Pazhouhandeh M, Kahrizi D. An overview of genome editing methods based on endonucleases. Mod Genet J 2020; 15(2): 75-92.
Ghaheri M, Kahrizi D, Bahrami G, Mohammadi-Motlagh HR. Study of gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni under various mannitol concentrations. Mol Biol Rep 2019; 46(1): 7-16.
Eruygur N, Ucar E, Akpulat HA, Shahsavari K, Safavi SM, Kahrizi D. In vitro antioxidant assessment, screening of enzyme inhibitory activities of methanol and water extracts and gene expression in Hypericum lydium. Mol Biol Rep 2019; 46(2): 2121-9.
Esmaeili F, Ghaheri M, Kahrizi D, Mansouri M, Safavi SM, Ghorbani T, Muhammadi S, Rahmanian E, Vaziri S. Effects of various glutamine concentrations on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni. Cell Mol Biol, 64(2): 1-5.

References

Prezzano KM, Ma SJ, Hermann GM. Stereotactic body radiation therapy for non-small cell lung cancer: A review. World J Clin Oncol 2019; 10(1):17-30.

Lembicz M, Gabryel P, Brajer-Luftmann B, Dyszkiewicz W, Batura-Gabryel H. Comorbidities with non-small cell lung cancer: Is there an interdisciplinary consensus needed to qualify patients for surgical treatment? Ann Thorac Med 2018; 13(2):101.

Di Benedetto F, Di Sandro S, De Ruvo N, Montalti R, Ballarin R, Guerrini GP, Spaggiari M, Guaraldi G, Gerunda G. First report on a series of HIV patients undergoing rapamycin monotherapy after liver transplantation. Transplant 2010; 89(6):733-8.

Turska"Kmieć A, Jankowska I, PawÅ‚owska J, KaliciÅ„ski P, Kawalec W, Tomyn M, Markiewicz M, Teisseyre J, Czubkowski P, Rć™kawek J, Socha J. Reversal of tacrolimus"related hypertrophic cardiomyopathy after conversion to rapamycin in a pediatric liver transplant recipient. Pediatr Transplant 2007; 11(3):319-23.

Alamo JM, Barrera L, Casado MD, Bernal C, Marin LM, Suarez G, Sanchez-Moreno L, Jimenez R, Suarez-Grau JM, Sousa JM, Cordero E. Efficacy, tolerance, and safety of mammalian target of rapamycin inhibitors as rescue immunosuppressants in liver transplantation. Transplant Proc 2009; 41: 6: 2181-2183).

Wang D, Gao L, Liu X, Yuan C, Wang G. Improved antitumor effect of ionizing radiation in combination with rapamycin for treating nasopharyngeal carcinoma. Oncol Lett 2017; 14(1):1105-8.

Liu PY, Zhang WB, Wang J, Shen YH, Wei YY. Inhibitory effect and significance of rapamycin on the mammalian target of rapamycin signaling pathway in osteosarcoma stem cells and osteosarcoma cells. Zhonghua Zhong Liu Za Zhi 2013; 35(3):175-80.

Bressac B, Puisieux A, Kew M, Volkmann M, Bozcall S, Mura JB, de la Monte S, Carlson R, Blum H, Wands J, Takahashi H. p53 mutation in hepatocellular carcinoma after aflatoxin exposure. Lancet 1991; 338(8779):1356-9.

Li L, Deng B, Xing G, Teng Y, Tian C, Cheng X, Yin X, Yang J, Gao X, Zhu Y, Sun Q. PACT is a negative regulator of p53 and essential for cell growth and embryonic development. Proc Natl Acad Sci 2007; 104(19):7951-6.

Kalemkerian GP, Ou X, Adil MR, Rosati R, Khoulani MM, Madan SK, Pettit GR. Activity of dolastatin 10 against small-cell lung cancer in vitro and in vivo: induction of apoptosis and bcl-2 modification. Cancer Chemother Pharmacol 1999; 43(6):507-15.

Miyake H, Hara S, Arakawa S, Kamidono S, Hara I. Overexpression of bcl"2 regulates sodium butyrate"and/or docetaxel"induced apoptosis in human bladder cancer cells both in vitro and in vivo. Int J Cancer 2001; 93(1):26-32.

Xiao ZM, Li LJ, Lu ZN, Li CY, Zheng JQ. Effects of extracellular Ca2+ influx and intracellular Ca2+ release on ethanol-induced cytoplasmic Ca2+ overload in cultured superior cervical ganglion neurons. Neurosci Lett 2005; 390(2):98-103.

Wang XZ, Zhang SJ, Chen YX, Chen ZX, Huang YH, Zhang LJ. Effects of platelet-derived growth factor and interleukin-10 on Fas/Fas-ligand and Bcl-2/Bax mRNA expression in rat hepatic stellate cells in vitro. World J Gastroenterol 2004; 10(18):2706.

Fuchs BC, Finger RE, Onan MC, Bode BP. ASCT2 silencing regulates mammalian target-of-rapamycin growth and survival signaling in human hepatoma cells. Am J Physiol Cell Physiol 2007; 293(1): C55-63.

Pencreach E, Guérin E, Nicolet C, Lelong-Rebel I, Voegeli AC, Oudet P, Larsen AK, Gaub MP, Guenot D. Marked activity of irinotecan and rapamycin combination toward colon cancer cells in vivo and in vitro is mediated through cooperative modulation of the mammalian target of rapamycin/hypoxia-inducible factor-1Î± axis. Clin Cancer Res 2009; 15(4):1297-307.

Jiang RY, Pei HL, Gu WD, Huang J, Wang ZG. Autophagic inhibitor attenuates rapamycin-induced inhibition of proliferation in cultured A549 lung cancer cells. Eur Rev Med Pharmacol Sci 2014; 18(6):806-10.

Webster NJ, Resnik JL, Reichart DB, Strauss B, Haas M, Seely BL. Repression of the insulin receptor promoter by the tumor suppressor gene product p53: a possible mechanism for receptor overexpression in breast cancer. Cancer Res 1996; 56(12):2781-8.

Li J, Jiang K, Zhao F. Icariin regulates the proliferation and apoptosis of human ovarian cancer cells through microRNA-21 by targeting PTEN, RECK and Bcl-2. Oncol Rep 2015; 33(6):2829-36.

Lee HJ, Wang CJ, Kuo HC, Chou FP, Jean LF, Tseng TH. Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of Bax/Bak and activation of JNK. Toxicol Appl Pharmacol 2005; 203(2):124-31.

Park KH, Kim SY, Gul R, Kim BJ, Jang KY, Chung HT, Sohn DH. Fatty acids ameliorate doxorubicin-induced intracellular Ca2+ increase and apoptosis in rat cardiomyocytes. Biol Pharm Bull 2008; 31(5):809-15.

Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, Reed JC. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Sci 1999; 284(5412):339-43.

Li M, Kondo T, Zhao QL, Li FJ, Tanabe K, Arai Y, Zhou ZC, Kasuya M. Apoptosis induced by cadmium in human lymphoma U937 cells through Ca2+-calpain and caspase-mitochondria-dependent pathways. J Biol Chem 2000; 275(50):39702-9.

Akbari F, Arminian A, Kahrizi D, Fazeli A, Ghaheri M. Effect of nitrogen sources on gene expression of Stevia rebaudiana (Bertoni) under in vitro conditions. Cell Mol Biol, 64(2): 11-16.

Akbarabadi A, Ismaili A, Kahrizi D, Firouzabadi FN. Validation of expression stability of reference genes in response to herbicide stress in wild oat (Avena ludoviciana). Cell Mol Biol 2018; 64(4). 113-118.

Ghorbani T, Kahrizi D, Saeidi M, Arji I. Effect of sucrose concentrations on Stevia rebaudiana Bertoni tissue culture and gene expression. Cell Mol Biol, 63(11): 32-36.

Ghaheri M, Adibrad E, Safavi SM, Kahrizi D, Soroush A, Muhammadi S, Ghorbani T, Sabzevari A, Ansarypour Z, Rahmanian E. Effects of life cycle and leaves location on gene expression and glycoside biosynthesis pathway in Stevia rebaudiana Bertoni. Cell Mol Biol, 64(2): 17-22.

Kahrizi D, Ghaheri M, Yari Z, Yari K, Bahraminejad S. Investigation of different concentrations of MS media effects on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni. Cell Mol Biol, 64(2): 23-27.

Hashempoor S, Ghaheri M, Kahrizi D, Kazemi N, Muhammadi S, Safavi SM, Ghorbani T, Rahmanian E, Heshmatpanaah M. Effects of different concentrations of mannitol on gene expression in Stevia rebaudiana Bertoni. Cell Mol Biol, 64(2): 28-31.

Kahrizi D, Ghari SM, Ghaheri M, Fallah F, Ghorbani T, Beheshti AA, Kazemi E, Ansarypour Z. Effect of KH2PO4 on gene expression, morphological and biochemical characteristics of stevia rebaudiana Bertoni under in vitro conditions. Cell Mol Biol, 63(7): 107-111.

Fallah F, Nokhasi F, Ghaheri M, Kahrizi D, Beheshti Ale AA, Ghorbani T, Kazemi E, Ansarypour Z. Effect of salinity on gene expression, morphological and biochemical characteristics of Stevia rebaudiana Bertoni under in vitro conditions. Cell Mol Biol 2017; 63(7): 102-106.

Bordbar M, Darvishzadeh R, Pazhouhandeh M, Kahrizi D. An overview of genome editing methods based on endonucleases. Mod Genet J 2020; 15(2): 75-92.

Ghaheri M, Kahrizi D, Bahrami G, Mohammadi-Motlagh HR. Study of gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni under various mannitol concentrations. Mol Biol Rep 2019; 46(1): 7-16.

Eruygur N, Ucar E, Akpulat HA, Shahsavari K, Safavi SM, Kahrizi D. In vitro antioxidant assessment, screening of enzyme inhibitory activities of methanol and water extracts and gene expression in Hypericum lydium. Mol Biol Rep 2019; 46(2): 2121-9.

Esmaeili F, Ghaheri M, Kahrizi D, Mansouri M, Safavi SM, Ghorbani T, Muhammadi S, Rahmanian E, Vaziri S. Effects of various glutamine concentrations on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni. Cell Mol Biol, 64(2): 1-5.

Author biographies is not available.