Rotenone isolated from Pachyrhizus erosus displays cytotoxicity and genotoxicity in K562 cells

Abstract

Pachyrhizus erosus (Fabaceae) is a herb commonly known as 'yam bean', which has been cultivated in México since pre-Columbian times for its edible tubers. The seeds are also known for their acaricidal and insecticidal properties due to rotenone and other isoflavonoid contents. Rotenone has exhibited cytotoxic activity against several human tumour cell lines; however, its mechanism of action is still not fully understood. In this study, we determined the cytotoxicity of rotenone isolated from P. erosus seeds on K562 human leukaemia cells. Rotenone exhibited significant cytotoxic activity (IC50 = 13.05 μM), as determined by the MTT assay. Three other isolated isoflavonoids were not cytotoxic. Rotenone genotoxicity was detected using the comet assay. Rotenone induced cell death, and caspase-3 activation as indicated by TUNEL assay, and immunocytofluorescence. Plasmid nicking assay indicated that rotenone does not interact directly with DNA.

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Rotenone isolated from Pachyrhizus
erosus displays cytotoxicity and
genotoxicity in K562 cells
Edgar A. Estrella-Parraa, Juan C. Gomez-Verjana, Ignacio
González-Sánchezab, Edgar Ricardo Vázquez-Martínezb, Edgar
Vergara-Castañedab, Marco A. Cerbónb, Dagoberto Alavez-Solanoc
& Ricardo Reyes-Chilpaa
a Departamento de Productos Naturales, Instituto de Química,
Universidad Nacional Autónoma de México, Mexico City, D.F.,
Mexico
b Departamento de Biología, Facultad de Química, Universidad
Nacional Autónoma de México, Mexico City, D.F., Mexico
c Departamento de Biología, Facultad de Ciencias, Universidad
Autónoma Benito Juárez de Oaxaca, Oaxaca, Mexico
Published online: 23 Jul 2014.
To cite this article: Edgar A. Estrella-Parra, Juan C. Gomez-Verjan, Ignacio González-Sánchez,
Edgar Ricardo Vázquez-Martínez, Edgar Vergara-Castañeda, Marco A. Cerbón, Dagoberto Alavez-
Solano & Ricardo Reyes-Chilpa (2014): Rotenone isolated from Pachyrhizus erosus displays
cytotoxicity and genotoxicity in K562 cells, Natural Product Research: Formerly Natural Product
Letters, DOI: 10.1080/14786419.2014.939081
To link to this article: http://dx.doi.org/10.1080/14786419.2014.939081
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SHORT COMMUNICATION
Rotenone isolated from Pachyrhizus erosus displays cytotoxicity and
genotoxicity in K562 cells
Edgar A. Estrella-Parra
a
, Juan C. Gomez-Verjan
a
, Ignacio Gonza
´lez-Sa
´nchez
ab
,
Edgar Ricardo Va
´zquez-Martı
´nez
b
, Edgar Vergara-Castan
˜eda
b
, Marco A. Cerbo
´n
b
,
Dagoberto Alavez-Solano
c
and Ricardo Reyes-Chilpa
a
*
a
Departamento de Productos Naturales, Instituto de Quı
´mica, Universidad Nacional Auto
´noma de Me
´xico,
Mexico City, D.F., Mexico;
b
Departamento de Biologı
´a, Facultad de Quı
´mica, Universidad Nacional
Auto
´noma de Me
´xico, Mexico City, D.F., Mexico;
c
Departamento de Biologı
´a, Facultad de Ciencias,
Universidad Auto
´noma Benito Jua
´rez de Oaxaca, Oaxaca, Mexico
(Received 19 March 2014; final version received 24 June 2014)
Pachyrhizus erosus (Fabaceae) is a herb commonly known as ‘yam bean’, which has
been cultivated in Me
´xico since pre-Columbian times for its edible tubers. The seeds
are also known for their acaricidal and insecticidal properties due to rotenone and other
isoflavonoid contents. Rotenone has exhibited cytotoxic activity against several human
tumour cell lines; however, its mechanism of action is still not fully understood. In this
study, we determined the cytotoxicity of rotenone isolated from P. erosus seeds on
K562 human leukaemia cells. Rotenone exhibited significant cytotoxic activity
(IC
50
¼13.05 mM), as determined by the MTT assay. Three other isolated
isoflavonoids were not cytotoxic. Rotenone genotoxicity was detected using the
comet assay. Rotenone induced cell death, and caspase-3 activation as indicated by
TUNEL assay, and immunocytofluorescence. Plasmid nicking assay indicated that
rotenone does not interact directly with DNA.
Keywords: Pachyrhizus erosus seeds; rotenone; K562 leukaemia cells; cytotoxicity;
apoptosis; caspase-3
1. Introduction
Pachyrhizus erosus (Fabaceae) is a tropical herb, native from Me
´xico and Central America. In
Me
´xico, P. erosus is known as ‘jicama’, and profusely cultivated since pre-Columbian time due
to it is edible subterranean tubercles. It has been introduced with the name ‘yam bean’ as a crop
into India, China and the USA, and used as a food ingredient (Alavez et al. 1998;Be
´jar et al.
2000). Rotenone is present in the seeds of P. erosus, and is well known as insect pesticide
(Alavez et al. 1998;Be
´jar et al. 2000). However, in the last 20 years rotenoids have acquired
pharmacological interest due to their antitumour properties (Kardono et al. 1990). Rotenone has
shown potent cytotoxic activity against several human tumour cell lines, such as breast (MCF-7;
IC
50
¼20 nM) and lung (A-549; IC
50
¼25 nM). Rotenone induces the production of reactive
oxygen species (ROS) which can damage DNA, RNA and proteins, eventually leading to cell
death (Gao et al. 2002; Li et al. 2003). In this study, we isolated four isoflavonoids from P.
erosus seeds and examined the cytotoxicity of such compounds against K562 human leukaemia
cells in vitro, and delve into the cellular and molecular mechanisms involved in the cytotoxicity
of rotenone to these cells.
q2014 Taylor & Francis
*Corresponding author. Email: chilpa@unam.mx
Natural Product Research, 2014
http://dx.doi.org/10.1080/14786419.2014.939081
Downloaded by [Unam - Centro De Nano Ciencias] at 08:47 31 July 2014

2. Results and discussion
2.1. Chemistry
The acetone extract of P. erosus seeds was subjected to column chromatography (Silica Gel 60)
as previously described (Alavez et al. 1998), obtaining four isoflavonoids: rotenone, dolineone,
12a-hydroxypachyrhizone and pachyrizine (Figure 1(A) – (D)), and their yields were 0.01%,
0.006%, 0.004% and 0.006%, respectively. The yield of the acetone extract was 0.35%.
2.2. Cytotoxic assay
The viability of K562 cells treated with the acetone extract from P. erosus seeds, or the isolated
compounds, was determined by the MTT assay. The P. erosus extract and rotenone displayed
IC
50
40.5 mg/mL and 13.05 mM, respectively. None of the other three isolated isoflavonoids
exhibited cytotoxic activity (maximum concentration evaluated was 150 mM). These results
agree with previous studies showing that rotenone displays cytotoxicity against several human
tumour cell lines (Sakurai et al. 2006). Also extracts containing rotenone from other Fabaceae
species, such as Antheroporum pierrei, had also displayed cytotoxic effect against human
tumour cell lines C38 and L1210 (Gao et al. 2011). Regarding structure/activity relationship,
rotenone was the only prenylated isoflavonoid. Interestingly, Cassidy and Setzer (2010)
established that prenylated isoflavonoids are more active against several cancer proteins than
non-prenylated isoflavonoids in a docking analysis (Cassidy & Setzer 2010).
AB
CD
Figure 1. Structure of isoflavonoids. (A) Rotenone, (B) dolineone, (C) 12a-hydroxypachyrhizone and (D)
pachyrhizine.
2E.A. Estrella-Parra et al.
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Figure 2. Genotoxicity induced by rotenone on K562 cells. Photomicrographs of K562 cells after (A) 1h, (B)
3 h and (C) 12h of rotenone treatment(13.05 mM). (D) Vehicle (DMSO; 12 h) and (E) positive control (H
2
O
2
).
Scale bar ¼100 mm. (F) Extent of tailmoment. Data shownare means from four independent experiments with
standard deviation. *p#0.05.
Figure 3. Plasmid nicking assay of rotenone. Electrophoretic pattern of pDEST26 DNA incubated with
different treatments. (A) Untreated pDEST26, (B) vehicle (DMSO), (C) rotenone (13.05 mM), (D) rotenone
(130 mM), (E) positive control (Eco RI endonuclease) and (F) Fenton’s reagent.
Natural Product Research 3
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2.3. Single-cell gel electrophoresis (comet assay)
The comet assay was performed to detect DNA damage in K562 cells. DNA damage induced by
rotenone (IC
50
¼13.05 mM) was evident after 1 h, peaked at 3 h and maintained after 12 h of
treatment (Figure 2). The positive control H
2
O
2
(10%) induced more than 80% of DNA damage
after 15 min. Comet assay indicated that rotenone induced genotoxicity 1 h after treatment on
K562 cells (Figure 2). It has been reported that other isoflavonoids, such as genistein, equol and
daidzein also induce DNA damage in human tumour cell lines as evaluated by this assay (Pool-
Zobel et al. 2000).
2.4. Plasmid nicking assay
In order to validate rotenone activity directly on DNA structure, pDEST26 DNA was treated with
two different concentrations of rotenone (13.05 and 130 mM). The DNA pattern of both treatments
was similar to the negative control (DMSO), but was different from the sample treated with
positive control (Fenton’s reagent and Eco RI endonuclease) (Figure 3). These findings indicated
that rotenone did not induce any structural effect on naked DNA, unlike Fenton’s reagent that
induces DNA random damage by hydroxyl radicals or as Eco RI endonuclease that produces DNA
cutting at specific nucleotides (one restriction site). This suggests that rotenone did not interact
directly with DNA (Figure 3). However, it has been reported that rotenone may induce DNA
damage through production of reactive oxygen (Radad et al. 2006; Deng et al. 2010). This supports
that the DNA damage observed in the comet assay may be due to the production of ROS.
2.5. TUNEL assay
TUNEL assay detects DNA fragmentation and suggests cell death by apoptosis. Other biochemical
changes that can be observed are chromatin condensation and nuclear fragmentation (Gonza
´lez-
Sa
´nchez et al. 2011). Observation by fluorescence microscope revealed small bright dots
representing chromatin condensation and/or nuclear fragmentation after 24 h of rotenone exposure
in K562 cells. Cells treated with vehicle (DMSO)did not show DNA fragmentation. Taxol was used
as positive control at 250 nM (Figure 4). Rotenone (13.05 mM) was positive in TUNEL at 24 h in
K562 cells (Figures 4 and 5), indicating that cell death might be due to apoptosis induction. The
rotenoid deguelin also induced TUNEL-positive cells and caspase-3 is activated in human Caucasian
colon adenocarcinoma (COLO 205) and colorectal carcinoma cell line (HCT116) (Kang et al. 2012).
2.6. Caspase-3 activity assay
Caspase-3 is an executioner caspase in apoptotic cell death and its activation is a crucial event
leading to apoptosis (Eom et al. 2010). The executioner caspase-3 was investigated to further
Figure 4. DNA fragmentation K562 cells determined by TUNEL assay. Cells were observed under
fluorescence microscope after 24 h of treatment. (A) Negative control (DMSO; 0.28%), (B) positive control
(taxol; 250 nM) and (C) rotenone (13.05 mM). Arrows show nuclei fragmentation. Scale bar ¼100 mm.
4E.A. Estrella-Parra et al.
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explore the cell death mechanism after rotenone treatment. It was observed that caspase-3 was
activated after rotenone treatment and taxol at 24 h. As expected, the cells treated with DMSO
were negative for active caspase-3 (Figure 5). These results are in accordance with previous
studies showing that rotenone (1 mM) induced the release of cytochrome cinto the cytosol and
caspase-dependent programmed cell death in SAS human tumour line (Lee et al. 2008).
Moreover, other antineoplastic compounds tested in K562 cells induced TUNEL-positive cells
and the activation of caspases (Gonza
´lez-Sa
´nchez et al. 2011).
3. Conclusion
Rotenone induces cytotoxicity via apoptosis in K562 cells through caspase-3 activation. These
results provide new insights into the molecular mechanisms involved in the rotenone-mediated
cytotoxicity.
Supplementary material
Supplementary material relating to this article is available online, alongside Materials and
methods.
Acknowledgements
We are also grateful to Marı
´a del Rocı
´o Patin
˜o Maya (IR), Marı
´a de los A
´ngeles Pen
˜a Gonza
´lez (RMN),
Francisco Javier Pe
´rez Flores (EM) for spectroscopical determination, to Alberto Javier Reyes Garcı
´a and
Araceli Zarate Aquino for botanical assistance. The authors have declared no conflict of interest.
Figure 5. Caspase-3 activated after rotenone treatment. Immunocytofluorescence analysis for activated
caspase-3 in K562 cells. Cells treated with negative control (DMSO; A, D, G), rotenone (B, E, H) and
positive control (taxol; C, F, I) at 24 h. DAPI-stained cells (A, B, C), anti-activated caspase-3 antibody (D,
E, F) and merge (G, H, I). Scale bar ¼100 mm.
Natural Product Research 5
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Funding
This research was supported by a grant IG-200513 from PAPIIT-DGAPA-UNAM. E.A. Estrella-Parra is
grateful to Posgrado en Ciencias Biolo
´gicas de la Universidad Nacional Autono
´ma de Me
´xico (PCB-
UNAM), and Consejo Nacional de Ciencia y Tecnologı
´a (CONACYT; 220355) for providing a scholarship
for Ph.D. studies. I. Gonza
´lez-Sa
´nchez was supported by a post-doctoral grant (DGAPA-UNAM).
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