β-sitosterol induces reactive oxygen species-mediated apoptosis in human hepatocellular carcinoma cell line

Document Type : Short communication


Department of Pharmacology, The Blue Lab, Molecular Medicine and Toxicology Division, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, India


Objective: It is of interest to investigate the anti-proliferative effect of β-sitosterol (BS) on human hepatocellular carcinoma (HepG2) cell line.
Materials and Methods: β-sitosterol treatments (0.6 and 1.2 mM/ml) were done in HepG2 and after 24 hr, cell viability was evaluated by MTT assay. Reactive oxygen species (ROS) accumulating potential of BS was assessed by dichloro-dihydro-fluorescein diacetate staining. Morphology related to apoptosis was investigated by acridine orange and ethidium bromide dual staining. Cytochrome c and caspase 3 expressions were evaluated by immunofluorescence and western blot analyses.
Results: β-sitosterol induced cytotoxicity (p<0.001) and intracellular ROS in HepG2 cells in a dose-dependent manner.BS treatments accumulated induced intracellular ROS accumulation which led to membrane damage and mitochondrial toxicity. At the molecular level, BS treatments induced cytochrome c release from mitochondria and enhanced the protein expressions (p<0.05 vs 0.6 mM/ml and p<0.001 vs 1.2 mM/ml) of both caspase 3 and cleaved caspase 3.
Conclusion: β-sitosterol induced ROS accumulation which plays a critical role in apoptosis via the intrinsic pathway in HepG2 cells. The present investigation paves the way for further in vivo studies.


Alvarez-Sala A, Attanzio A, Tesoriere L,
Garcia-Llatas G, Barberá R, Cilla A. 2019.
Apoptotic effect of a phytosterol-ingredient
and its main phytosterol (β-sitosterol) in
human cancer cell lines. Int J Food Sci Nutr,
70: 323-334.
Solai Prakash AK, Devaraj E. 2019. Cytotoxic
potentials of S. cumini methanolic seed
kernel extract in human hepatoma HepG2
cells. Environ Toxicol, 34: 1313-1319.
Awad AB, Barta SL, Fink CS, Bradford PG.
2008. Beta-sitosterol enhances tamoxifen
effectiveness on breast cancer cells by
affecting ceramide metabolism. Mol Nutr
Food Res, 52: 419-426.
Awad AB, Fink CS, Williams H, Kim U. 2001.
In vitro and in vivo (SCID mice) effects of
phytosterols on the growth and
dissemination of human prostate cancer PC3 cells. Eur J Cancer Prev, 10: 507-513.
Bacchetti T, Masciangelo S, Bicchiega V,
Bertoli E, Ferretti G. 2011. Phytosterols,
phytostanols and their esters: from natural to
functional foods. Med J Nutrition Metab, 4:
Baskar AA, Al Numair KS, Gabriel Paulraj M,
Alsaif MA, Muamar MA, Ignacimuthu S.
2012. β-sitosterol prevents lipid
peroxidation and improves antioxidant
status and histoarchitecture in rats with 1,2-
dimethylhydrazine-induced colon cancer. J
Med Food, 15: 335-343.
Baskar AA, Ignacimuthu S, Paulraj GM, Al
Numair KS. 2010. Chemopreventive
potential of beta-Sitosterol in experimental
colon cancer model-an in vitro and In vivo
study. BMC Complement Altern Med, 10:
Byczkowska A, Kunikowska A, Kaźmierczak
A. 2013. Determination of ACC-induced
cell-programmed death in roots of Vicia
faba ssp. minor seedlings by acridine orange
and ethidium bromide staining.
Protoplasma, 250: 121-128.
Cao ZQ, Wang XX, Lu L, Xu JW, Li XB,
Zhang GR, Ma ZJ, Shi AC, Wang Y, Song
YJ. 2019. β-sitosterol and gemcitabine
exhibit synergistic anti-pancreatic cancer
activity by modulating apoptosis and
inhibiting epithelial-mesenchymal transition
by deactivating Akt/GSK-3β signaling.
Front Pharmacol, 9: 1525.
Chirumbolo S, Bjørklund G, Lysiuk R, Vella A,
Lenchyk L, Upyr T. 2018. Targeting cancer
with phytochemicals via their fine tuning of
the cell survival signaling pathways. Int J
Mol Sci, 19: 3568.
Choi YH, Kong KR, Kim YA, Jung KO, Kil JH,
Rhee SH, Park KY. 2003. Induction of Bax
and activation of caspases during betasitosterol-mediated apoptosis in human
colon cancer cells. Int J Oncol, 23: 1657-
Devaraj E, Roy A, Royapuram Veeraragavan
G, Magesh A, Varikalam Sleeba A,
Arivarasu L, Marimuthu Parasuraman B.
2020. β-Sitosterol attenuates carbon
tetrachloride-induced oxidative stress and
chronic liver injury in rats. Naunyn
Schmiedebergs Arch Pharmacol, 393: 1067-
Ezhilarasan D, Apoorva VS, Ashok Vardhan N.
2019. Syzygium cumini extract induced
reactive oxygen species-mediated apoptosis
in human oral squamous carcinoma cells. J
Oral Pathol Med, 48: 115-121.
Ezhilarasan D. 2018. Herbal therapy for cancer.
In: Prakash Srinivasan, Timiri Shanmugam.,
editors. Understanding Cancer Therapies,
CRC Press, pp. 129–166.
Gheena S, Ezhilarasan D. 2019. Syringic acid
triggers reactive oxygen species-mediated
cytotoxicity in HepG2 cells. Hum Exp
Toxicol, 38: 694-702.
Hartke J, Johnson M, Ghabril M. 2017. The
diagnosis and treatment of hepatocellular
carcinoma. Semin Diagn Pathol, 34: 153-
Hong SH, Cha HJ, Hwang-Bo H, Kim MY,
Kim SY, Ji SY, Cheong J, Park C, Lee H,
Kim GY, Moon SK, Yun SJ, Chang YC,
Kim WJ, Choi YH. 2019. Anti-proliferative
and pro-apoptotic effects of licochalcone A
through ROS-mediated cell cycle arrest and
apoptosis in human bladder cancer cells. Int
J Mol Sci, 20: 3820.
Hosseini A, Ghorbani A. 2015. Cancer therapy
with phytochemicals: evidence from clinical
studies. Avicenna J Phytomed, 5: 84-97.
Jan R, Chaudhry GE. 2019. Understanding
apoptosis and apoptotic pathways targeted
cancer therapeutics. Adv Pharm Bull, 9:
Jiang JF, Lao YC, Yuan BH, Yin J, Liu X, Chen
L, Zhong JH. 2017. Treatment of
hepatocellular carcinoma with portal vein
tumor thrombus: advances and challenges.
Oncotarget, 8: 33911-33921.
β-sitosterol induces cytotoxicity in HepG2 cells
AJP, Vol. 11, No. 6, Nov-Dec 2021 549
Jiang L, Zhao X, Xu J, Li C, Yu Y, Wang W,
Zhu L. 2019. The protective effect of dietary
Phytosterols on cancer risk: A systematic
meta-analysis. J Oncol, 2019: 7479518.
Jones PJ, AbuMweis SS. 2009. Phytosterols as
functional food ingredients: linkages to
cardiovascular disease and cancer. Curr
Opin Clin Nutr Metab Care, 12: 147-151.
Kasibhatla S, Amarante-Mendes GP, Finucane
D, Brunner T, Bossy-Wetzel E, Green DR.
2006. Acridine orange/ethidium bromide
(AO/EB) staining to detect apoptosis. CSH
Protoc, 2006: pdb.prot4493.
Kazłowska K, Lin HT, Chang SH, Tsai GJ.
2013. In vitro and in vivo anticancer effects
of sterol fraction from red algae porphyra
dentata. Evid Based Complement Alternat
Med, 2013: 493869.
Lakshmi T, Ezhilarasan D, Vijayaragavan R,
Bhullar SK, Rajendran R. 2017. Acacia
catechu ethanolic bark extract induces
apoptosis in human oral squamous
carcinoma cells. J Adv Pharm Technol Res,
8: 143-149.
Li S, Jiang Z, Chai W, Xu Y, Wang Y. 2019.
Autophagy activation alleviates
nonylphenol-induced apoptosis in cultured
cortical neurons. Neurochem Int, 122: 73-
Li X, Wu Q, Bu M, Hu L, Du WW, Jiao C, Pan
H, Sdiri M, Wu N, Xie Y, Yang BB. 2016.
Ergosterol peroxide activates Foxo3-
mediated cell death signaling by inhibiting
AKT and c-Myc in human hepatocellular
carcinoma cells. Oncotarget, 7: 33948-
Liu K, Liu PC, Liu R, Wu X. 2015. Dual
AO/EB staining to detect apoptosis in
osteosarcoma cells compared with flow
cytometry. Med Sci Monit Basic Res, 21:
Misawa E, Tanaka M, Nomaguchi K,
Nabeshima K, Yamada M, Toida T,
Iwatsuki K. 2012. Oral ingestion of aloe
vera phytosterols alters hepatic gene
expression profiles and ameliorates obesityassociated metabolic disorders in zucker
diabetic fatty rats. J Agric Food Chem, 60:
Pfeffer CM, Singh ATK. 2018. Apoptosis: A
target for anticancer therapy. Int J Mol Sci,
19: E448.
PonselviInduja M, Ezhilarasan D, Ashok
Vardhan N. 2018. Evolvulus alsinoides
methanolic extract triggers apoptosis in
HepG2 cells. Avicenna J Phytomed, 8: 504-
Kathiswar R, Ezhilarasan D, Rajeshkumar S.
2020. β-Sitosterol-assisted silver
nanoparticles activates Nrf2 and triggers
mitochondrial apoptosis via oxidative stress
in human hepatocellular cancer cell line. J
Biomed Mater Res A, 108: 1899-1908.
Rajavel T, Packiyaraj P, Suryanarayanan V,
Singh SK, Ruckmani K, Pandima Devi K.
2018. β-sitosterol targets Trx/Trx1 reductase
to induce apoptosis in A549 cells via ROS
mediated mitochondrial dysregulation and
p53 activation. Sci Rep, 8: 2071.
Rajavel T, Mohankumar R, Archunan G,
Ruckmani K, Devi KP. 2017. Beta sitosterol
and Daucosterol (phytosterols identified in
Grewia tiliaefolia) perturbs cell cycle and
induces apoptotic cell death in A549 cells.
Sci Rep, 7: 3418.
Ramprasath VR, Awad AB. 2015. Role of
phytosterols in cancer prevention and
treatment. J AOAC Int, 98: 735-738.
Rayan A, Raiyn J, Falah M. 2017. Nature is the
best source of anticancer drugs: Indexing
natural products for their anticancer
bioactivity. PLoS One, 12: e0187925.
Rohit Singh T, Ezhilarasan D. 2019. Ethanolic
extract of Lagerstroemia Speciosa (L.)
Pers., induces apoptosis and cell cycle arrest
in HepG2 cells. Nutr Cancer, 72: 146-156.
Sharmila R, Sindhu G. 2017. Modulation of
angiogenesis, proliferative response and
apoptosis by BS in rat model of renal
carcinogenesis. Indian J Clin Biochem, 32:
Shebi S, Ezhilarasan D, Thomas J,
Chandrasekaran N, Mukherjee A. 2019.
Gracilaria foliifera (Forssk.) Børgesen
ethanolic extract triggers apoptosis via
activation of p53 expression in HepG2 cells.
Phcog Mag, 15: 259-263.
Shin EJ, Choi HK, Sung MJ, Park JH, Chung
MY, Chung S, Hwang JT. 2018. Antitumour effects of beta-sitosterol are
mediated by AMPK/PTEN/HSP90 axis in
AGS human gastric adenocarcinoma cells
and xenograft mouse models. Biochem
Pharmacol, 152: 60-70.
Siegel RL, Miller KD, Jemal A. 2016. Cancer
statistics, 2016.CA Cancer J Clin, 66: 7-30.
Suh JK, Lee J, Lee JH, Shin S, Tchoe HJ, Kwon
JW. 2018. Risk factors for developing liver
cancer in people with and without liver
disease. PLoS One, 13: e0206374.
Ditty et al.
AJP, Vol. 11, No. 6, Nov-Dec 2021 550
Suttiarporn P, Chumpolsri W, Mahatheeranont
S, Luangkamin S, Teepsawang S,
Leardkamolkarn V. 2015. Structures of
phytosterols and triterpenoids with potential
anti-cancer activity in bran of black nonglutinous rice. Nutrients, 7: 1672-1687.
Yang C, Chen ZY, Wong SL, Liu J, Liang YT,
Lau CW, Lee HK, Huang Y, Tsang SY.
2013. β-Sitosterol oxidation products
attenuate vasorelaxation by increasing
reactive oxygen species and
cyclooxygenase-2. Cardiovasc Res, 97: 520-
Yang Y, Karakhanova S, Hartwig W, D'Haese
JG, Philippov PP, Werner J, Bazhin AV.
2016. Mitochondria and mitochondrial ROS
in cancer: Novel targets for anticancer
therapy. J Cell Physiol, 231: 2570-2581.
Zaloga GP. 2015. Phytosterols, lipid
administration, and liver disease during
parenteral nutrition. JPEN J Parenter Enteral
Nutr, 39: 39S-60S.