Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

At a glance: The STEP trials

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.
ADVANCED SEARCH
Log in
Top
Journal of Experimental & Clinical Cancer Research
Published in: Journal of Experimental & Clinical Cancer Research 1/2016

Open Access 01-12-2016 | Research

A piperazidine derivative of 23-hydroxy betulinic acid induces a mitochondria-derived ROS burst to trigger apoptotic cell death in hepatocellular carcinoma cells

Authors: Nan Yao, Ying-jie Li, Yu-he Lei, Nan Hu, Wei-Min Chen, Zhe Yao, Miao Yu, Jun-shan Liu, Wen-cai Ye, Dong-mei Zhang

Published in: Journal of Experimental & Clinical Cancer Research | Issue 1/2016

Login to get access

Abstract

Background

Elevated production of reactive oxygen species (ROS) and an altered redox state have frequently been observed in hepatocellular carcinoma (HCC); therefore, selective killing of HCC cells by chemotherapeutic agents that stimulate ROS generation or impair antioxidant systems may be a feasible approach in HCC chemotherapy. Recently, betulinic acid and its derivatives have attracted attention because they showed anti-cancer effects via a ROS- and mitochondria-related mechanism. However, the source of ROS overproduction and the role of mitochondria were poorly identified, and the weak in vivo antitumour activity of these compounds limits their development as drugs.

Methods

Cytotoxicity was detected using MTT assays. In vivo anti-HCC effects were assessed using nude mice bearing HepG2 tumour xenografts. Cell cycle analysis, apoptosis rate and mitochondrial membrane potential were measured by flow cytometry. ROS production was detected using a microplate reader or a fluorescence microscope. Changes in gene and protein levels were measured by RT-PCR and western blotting, respectively. Other assays were performed using related detection kits.

Results

B5G9, a piperazidine derivative of 23-hydroxy betulinic acid (23-HBA), showed excellent in vivo anti-HCC effects, with a tumour growth inhibitory rate of greater than 80%, and no significant side effects. B5G9 stimulated the production of ROS, which were derived from the mitochondria, but it had no effect on various other antioxidant systems. Moreover, B5G9 induced mitochondrial dysfunction, which was characterized by morphological changes, membrane potential collapse, membrane permeabilization, and decreases in the O2 consumption rate and ATP production. Furthermore, mtDNA-depleted ρ0 HepG2 cells were less sensitive to B5G9 treatment than wt HepG2 cells, indicating the importance of mitochondria in B5G9-induced cell death.

Conclusion

We discovered a piperazidine derivative of 23-HBA, B5G9, with excellent anti-HCC effects both in vivo and in vitro and no obvious toxic effects. The underlying mechanism was associated with mitochondria-derived ROS overproduction, and mitochondria played essential roles in B5G9-induced cell death. This study identified a potential agent for anti-HCC therapy and elucidated the mitochondria-related mechanism of BA and its derivatives.
Appendix
Available only for authorised users
Literature
1.
Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 2010;127:2893–917.CrossRefPubMed
2.
Leung T, Johnson PJ. Systemic therapy for hepatocellular carcinoma. Cancer J. 2013;2:514–20.
3.
4.
Wang X, Wang N, Cheung F, Lao L, Li C, Feng Y. Chinese medicines for prevention and treatment of human hepatocellular carcinoma: current progress on pharmacological actions and mechanisms. J Integr Med. 2015;13:142–64.CrossRefPubMed
5.
Devasagayam TP, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS, Lele RD. Free radicals and antioxidants in human health: current status and future prospects. J Assoc Physicians India. 2004;52:794–804.PubMed
6.
Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol. 2002;192:1–15.CrossRefPubMed
7.
Szatrowski TP, Nathan CF. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res. 1991;51:794–8.PubMed
8.
Toyokuni S, Okamoto K, Yodoi J, Hiai H. Persistent oxidative stress in cancer. FEBS Lett. 1995;358:1–3.CrossRefPubMed
9.
10.
11.
Nickel A, Kohlhaas M, Maack C. Mitochondrial reactive oxygen species production and elimination. J Mol Cell Cardiol. 2014;73:26–33.CrossRefPubMed
12.
13.
Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007;87:245–313.CrossRefPubMed
14.
Paravicini TM, Touyz RM. NADPH oxidases, reactive oxygen species, and hypertension: clinical implications and therapeutic possibilities. Diabetes Care. 2008;31 Suppl 2:S170–180.CrossRefPubMed
15.
Trachootham D, Zhou Y, Zhang H, Demizu Y, Chen Z, Pelicano H, Chiao PJ, Achanta G, Arlinghaus RB, Liu J, Huang P. Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate. Cancer Cell. 2006;10:241–52.CrossRefPubMed
16.
Lin J, Zahurak M, Beer TM, Ryan CJ, Wilding G, Mathew P, Morris M, Callahan JA, Gordon G, Reich SD, et al. A non-comparative randomized phase II study of 2 doses of ATN-224, a copper/zinc superoxide dismutase inhibitor, in patients with biochemically recurrent hormone-naive prostate cancer. Urol Oncol. 2013;31:581–8.CrossRefPubMed
17.
You BR, Shin HR, Park WH. PX-12 inhibits the growth of A549 lung cancer cells via G2/M phase arrest and ROS-dependent apoptosis. Int J Oncol. 2014;44:301–8.PubMed
18.
Cichewicz RH, Kouzi SA. Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection. Med Res Rev. 2004;24:90–114.CrossRefPubMed
19.
Fulda S, Kroemer G. Targeting mitochondrial apoptosis by betulinic acid in human cancers. Drug Discov Today. 2009;14:885–90.CrossRefPubMed
20.
Tan Y, Yu R, Pezzuto JM. Betulinic acid-induced programmed cell death in human melanoma cells involves mitogen-activated protein kinase activation. Clin Cancer Res. 2003;9:2866–75.PubMed
21.
Chintharlapalli S, Papineni S, Ramaiah SK, Safe S. Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Res. 2007;67:2816–23.CrossRefPubMed
22.
Hsu TI, Wang MC, Chen SY, Huang ST, Yeh YM, Su WC, Chang WC, Hung JJ. Betulinic acid decreases specificity protein 1 (Sp1) level via increasing the sumoylation of sp1 to inhibit lung cancer growth. Mol Pharmacol. 2012;82:1115–28.CrossRefPubMed
23.
Pandey MK, Sung B, Aggarwal BB. Betulinic acid suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase SHP-1 in human multiple myeloma cells. Int J Cancer. 2010;127:282–92.PubMedPubMedCentral
24.
Lan P, Wang J, Zhang DM, Shu C, Cao HH, Sun PH, Wu XM, Ye WC, Chen WM. Synthesis and antiproliferative evaluation of 23-hydroxybetulinic acid derivatives. Eur J Med Chem. 2011;46:2490–502.CrossRefPubMed
25.
Zhang H, Wang Y, Zhu P, Liu J, Xu S, Yao H, Jiang J, Ye W, Wu X, Xu J. Design, synthesis and antitumor activity of triterpenoid pyrazine derivatives from 23-hydroxybetulinic acid. Eur J Med Chem. 2015;97:235–44.CrossRefPubMed
26.
Fulda S, Debatin KM. Betulinic acid induces apoptosis through a direct effect on mitochondria in neuroectodermal tumors. Med Pediatr Oncol. 2000;35:616–8.CrossRefPubMed
27.
Wick W, Grimmel C, Wagenknecht B, Dichgans J, Weller M. Betulinic acid-induced apoptosis in glioma cells: A sequential requirement for new protein synthesis, formation of reactive oxygen species, and caspase processing. J Pharmacol Exp Ther. 1999;289:1306–12.PubMed
28.
Cheng Y, Wen-Cai YE, Shi JM, Zhang K, Yan-Qing L, Yao XS, Kurihara H. Role of reactive oxygen species in 23-hydroxybetulinic acid-induced apoptosis in LoVo cells. Chin J Pathophysiol. 2007;23:772–5.
29.
Bernardo TC, Cunha-Oliveira T, Serafim TL, Holy J, Krasutsky D, Kolomitsyna O, Krasutsky P, Moreno AM, Oliveira PJ. Dimethylaminopyridine derivatives of lupane triterpenoids cause mitochondrial disruption and induce the permeability transition. Bioorg Med Chem. 2013;21:7239–49.CrossRefPubMed
30.
Zhang DM, Xu HG, Wang L, Li YJ, Sun PH, Wu XM, Wang GJ, Chen WM, Ye WC. Betulinic acid and its derivatives as potential antitumor agents. Med Res Rev. 2015;35:1127–55.CrossRefPubMed
31.
Zhang H, Zhu P, Liu J, Yang X, Xu S, Yao H, Jiang J, Ye W, Wu X, Xu J. Synthesis and antitumor activity of novel 3-oxo-23-hydroxybetulinic acid derivatives. Eur J Med Chem. 2014;87:159–67.CrossRefPubMed
32.
Zhang H, Zhu P, Liu J, Lin Y, Yao H, Jiang J, Ye W, Wu X, Xu J. Synthesis, in vitro and in vivo antitumor activity of pyrazole-fused 23-hydroxybetulinic acid derivatives. Bioorg Med Chem Lett. 2015;25:728–32.CrossRefPubMed
33.
Zhang H, Li F, Zhu P, Liu J, Yao H, Jiang J, Ye W, Wu X, Xu J. Synthesis and biological evaluation of oxygen-containing heterocyclic ring-fused 23-hydroxybetulinic acid derivatives as antitumor agents. Chem Biol Drug Des. 2015;86:424–31.CrossRefPubMed
34.
Liu SJ, Sun YM, Tian DF, He YC, Zeng L, He Y, Ling CQ, Sun SH. Downregulated NM23-H1 expression is associated with intracranial invasion of nasopharyngeal carcinoma. Br J Cancer. 2008;98:363–9.CrossRefPubMedPubMedCentral
35.
Lei X, Chen M, Nie Q, Hu J, Zhuo Z, Yiu A, Chen H, Xu N, Huang M, Ye K, et al. In vitro and in vivo antiangiogenic activity of desacetylvinblastine monohydrazide through inhibition of VEGFR2 and Axl pathways. Am J Cancer Res. 2016;6:843–58.PubMedPubMedCentral
36.
Du Y, Sun H, Chen H, Wang C, Li Y, Wang X, Sun G. Reference intervals for routine blood tests in 468 healthy Mongolian children. Clin Lab. 2015;61:1763–7.PubMed
37.
Zhao X, Pan YP, Zhang DM, Liu JB. [Study of serological biochemistry index of chronic periodontitis]. Hua Xi Kou Qiang Yi Xue Za Zhi. 2010;28:584–6. 598.PubMed
38.
Park SY, Chang I, Kim JY, Kang SW, Park SH, Singh K, Lee MS. Resistance of mitochondrial DNA-depleted cells against cell death: role of mitochondrial superoxide dismutase. J Biol Chem. 2004;279:7512–20.CrossRefPubMed
39.
Kim JY, Kim YH, Chang I, Kim S, Pak YK, Oh BH, Yagita H, Jung YK, Oh YJ, Lee MS. Resistance of mitochondrial DNA-deficient cells to TRAIL: role of Bax in TRAIL-induced apoptosis. Oncogene. 2002;21:3139–48.CrossRefPubMed
40.
Adrain C, Creagh EM, Martin SJ. Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2. EMBO J. 2001;20:6627–36.CrossRefPubMedPubMedCentral
41.
42.
43.
Zuo L, Zhou T, Pannell BK, Ziegler AC, Best TM. Biological and physiological role of reactive oxygen species--the good, the bad and the ugly. Acta Physiol (Oxf). 2015;214:329–48.CrossRef
44.
Methy D, Bertrand N, Prigent-Tessier A, Stanimirovic D, Beley A, Marie C. Differential MnSOD and HO-1 expression in cerebral endothelial cells in response to sublethal oxidative stress. Brain Res. 2004;1003:151–8.CrossRefPubMed
45.
Liu YX, Wang J, Guo J, Wu J, Lieberman HB, Yin Y. DUSP1 is controlled by p53 during the cellular response to oxidative stress. Mol Cancer Res. 2008;6:624–33.CrossRefPubMed
46.
Liu X, Nie S, Huang D, Xie M. Nonylphenol regulates cyclooxygenase-2 expression via Ros-activated NF-kappaB pathway in sertoli TM4 cells. Environ Toxicol. 2015;30:1144–52.CrossRefPubMed
47.
Gao Z, Zhang H, Liu J, Lau CW, Liu P, Chen ZY, Lee HK, Tipoe GL, Ho HM, Yao X, Huang Y. Cyclooxygenase-2-dependent oxidative stress mediates palmitate-induced impairment of endothelium-dependent relaxations in mouse arteries. Biochem Pharmacol. 2014;91:474–82.CrossRefPubMed
48.
Ogura J, Kuwayama K, Sasaki S, Kaneko C, Koizumi T, Yabe K, Tsujimoto T, Takeno R, Takaya A, Kobayashi M, et al. Reactive oxygen species derived from xanthine oxidase interrupt dimerization of breast cancer resistance protein, resulting in suppression of uric acid excretion to the intestinal lumen. Biochem Pharmacol. 2015;97:89–98.CrossRefPubMed
49.
Thornber K, Colomba A, Ceccato L, Delsol G, Payrastre B, Gaits-Iacovoni F. Reactive oxygen species and lipoxygenases regulate the oncogenicity of NPM-ALK-positive anaplastic large cell lymphomas. Oncogene. 2009;28:2690–6.CrossRefPubMed
50.
Beak SM, Lee YS, Kim JA. NADPH oxidase and cyclooxygenase mediate the ultraviolet B-induced generation of reactive oxygen species and activation of nuclear factor-kappaB in HaCaT human keratinocytes. Biochimie. 2004;86:425–9.CrossRefPubMed
51.
Hrycay EG, Bandiera SM. Involvement of cytochrome P450 in reactive oxygen species formation and cancer. Adv Pharmacol. 2015;74:35–84.CrossRefPubMed
52.
53.
Yang J, Qiu B, Li X, Zhang H, Liu W. p53-p66(shc)/miR-21-Sod2 signaling is critical for the inhibitory effect of betulinic acid on hepatocellular carcinoma. Toxicol Lett. 2015;238:1–10.CrossRefPubMed
54.
Li N, Ragheb K, Lawler G, Sturgis J, Rajwa B, Melendez JA, Robinson JP. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem. 2003;278:8516–25.CrossRefPubMed
55.
Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, Schumacker PT. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. J Biol Chem. 2000;275:25130–8.CrossRefPubMed
56.
Bermudez O, Pages G, Gimond C. The dual-specificity MAP kinase phosphatases: critical roles in development and cancer. Am J Physiol Cell Physiol. 2010;299:C189–202.CrossRefPubMed
57.
Lin YW, Chuang SM, Yang JL. ERK1/2 achieves sustained activation by stimulating MAPK phosphatase-1 degradation via the ubiquitin-proteasome pathway. J Biol Chem. 2003;278:21534–41.CrossRefPubMed
58.
Calvisi DF, Pinna F, Meloni F, Ladu S, Pellegrino R, Sini M, Daino L, Simile MM, De Miglio MR, Virdis P, et al. Dual-specificity phosphatase 1 ubiquitination in extracellular signal-regulated kinase-mediated control of growth in human hepatocellular carcinoma. Cancer Res. 2008;68:4192–200.CrossRefPubMed
59.
Mullauer FB, Kessler JH, Medema JP. Betulinic acid induces cytochrome c release and apoptosis in a Bax/Bak-independent, permeability transition pore dependent fashion. Apoptosis. 2009;14:191–202.CrossRefPubMed
60.
Potze L, Mullauer FB, Colak S, Kessler JH, Medema JP. Betulinic acid-induced mitochondria-dependent cell death is counterbalanced by an autophagic salvage response. Cell Death Dis. 2014;5:e1169.CrossRefPubMed
61.
Potze L, Di Franco S, Grandela C, Pras-Raves ML, Picavet DI, van Veen HA, van Lenthe H, Mullauer FB, van der Wel NN, Luyf A, et al. Betulinic acid induces a novel cell death pathway that depends on cardiolipin modification. Oncogene. 2016;35:427–37.CrossRefPubMed
62.
Chen Y, Sun R, Wang B. Monolayer behavior of binary systems of betulinic acid and cardiolipin: thermodynamic analyses of Langmuir monolayers and AFM study of Langmuir-Blodgett monolayers. J Colloid Interface Sci. 2011;353:294–300.CrossRefPubMed
Metadata
Title
A piperazidine derivative of 23-hydroxy betulinic acid induces a mitochondria-derived ROS burst to trigger apoptotic cell death in hepatocellular carcinoma cells
Authors
Nan Yao
Ying-jie Li
Yu-he Lei
Nan Hu
Wei-Min Chen
Zhe Yao
Miao Yu
Jun-shan Liu
Wen-cai Ye
Dong-mei Zhang
Publication date
01-12-2016
Publisher
BioMed Central
Published in
Journal of Experimental & Clinical Cancer Research / Issue 1/2016
Electronic ISSN: 1756-9966
DOI
https://doi.org/10.1186/s13046-016-0457-1

Other articles of this Issue 1/2016

Journal of Experimental & Clinical Cancer Research 1/2016 Go to the issue

Research

Flow cytometry remission by Ig light chains ratio is a powerful marker of outcome in multiple myeloma after tandem autologous transplant: a real-life study

Research

Long non-coding RNA XIST regulates gastric cancer progression by acting as a molecular sponge of miR-101 to modulate EZH2 expression

Research

Evaluation of intraarterial and intravenous cisplatin chemotherapy in the treatment of metastatic osteosarcoma using an orthotopic xenograft mouse model

Review

Dysregulation of microRNA biogenesis in cancer: the impact of mutant p53 on Drosha complex activity

Research

Up-regulation of long non-coding RNA PANDAR is associated with poor prognosis and promotes tumorigenesis in bladder cancer

Research

Efficacy and safety of aflibercept in in vitro and in vivo models of retinoblastoma
Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras
Prof. Fabrice Barlesi
Developed by: Springer Medicine
Image Credits