Cistanche tubulosa induces reactive oxygen species-mediated apoptosis of primary and metastatic human colon cancer cells

Abstract

Colon cancer is the third most common cancer worldwide. Conventional therapies have shown moderate efficacy with severe adverse effects, therefore there is an urgent need for safer alternatives. In this study, Cistanche tubulosa, local name Thanoon, was considered as a potential phytotherapeutic strategy because of its known high therapeutic potential in traditional medicine and wide abundance in the Middle East region. Bioactive compounds were extracted from powdered Cistanche tubulosa and tested for their anticancer properties against four colon cancer cell lines including two derived from primary tumor (CaCo2 and HCT116) and two derived from metastatic site (LoVo and SW620). Effect of Cistanche tubulosa on induction of apoptosis and cellular redox homeostasis were also investigated. Cistanche tubulosa exhibited a concentration and time-dependent inhibition of proliferation of all tested cancer cell lines by more than 60% upon 72 hours treatment with 1 mg/mL of crude extract. Inhibition of proliferation was marked by induction of apoptosis, intracellular reactive oxygen species production and mitochondrial superoxides. This data suggest that Cistanche tubulosa is a promising candidate for additive anti-colon cancer therapy. This is the first study showing anticancer bioactivity of Cistanche tubulosa against colon cancer cells.

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© 2016 Pachacama Al-Menhali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-
ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
Journal of Applied Pharmaceutical Science Vol. 7 (05), pp. 039-045, May, 2017
Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2017.70507
ISSN 2231-3354
Cistanche tubulosa induces reactive oxygen species-mediated apoptosis
of primary and metastatic human colon cancer cells
Afnan Saleh Al-Menhali1, Safya Ali Jameela1, Aishah A. Latiff1, Mohamed A. Elrayess2, Mohammed Alsayrafi1,
Morana Jaganjac1*
1Anti Doping Lab Qatar, Toxicology and Multipurpose, Doha, Qatar.
2Anti Doping Lab Qatar, Life Science Research, Doha, Qatar.
ARTICLE INFO
ABSTRACT
Article history:
Received on: 01/11/2016
Accepted on: 07/01/2017
Available online: 30/05/2017
Colon cancer is the third most common cancer worldwide. Conventional therapies have shown moderate
efficacy with severe adverse effects, therefore there is an urgent need for safer alternatives. In this study,
Cistanche tubulosa, local name Thanoon, was considered as a potential phytotherapeutic strategy because of its
known high therapeutic potential in traditional medicine and wide abundance in the Middle East region.
Bioactive compounds were extracted from powdered Cistanche tubulosa and tested for their anticancer
properties against four colon cancer cell lines including two derived from primary tumor (CaCo2 and HCT116)
and two derived from metastatic site (LoVo and SW620). Effect of Cistanche tubulosa on induction of apoptosis
and cellular redox homeostasis were also investigated. Cistanche tubulosa exhibited a concentration and time-
dependent inhibition of proliferation of all tested cancer cell lines by more than 60% upon 72 hours treatment
with 1 mg/mL of crude extract. Inhibition of proliferation was marked by induction of apoptosis, intracellular
reactive oxygen species production and mitochondrial superoxides. This data suggest that Cistanche tubulosa is
a promising candidate for additive anti-colon cancer therapy. This is the first study showing anticancer
bioactivity of Cistanche tubulosa against colon cancer cells.
Abbreviations: 7-AAD, 7-Amino-Actinomycin D; CTE, Cistanche tubulosa extract; EMEM, Eagle's Minimum
Essential Medium; FBS, fetal bovine serum; DCF, 2,7-dichlorofluorescein; DCFH-DA, 2,7-
dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO,
dimethylsulfoxide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium; PE, Phycoerythrin; RFU,
relative fluorescence units; ROS, reactive oxygen species
Key words:
Cistanche tubulosa, colon
cancer, redox homeostasis,
anticancer bioactivity.
INTRODUCTION
Colorectal cancer is one of the most common cancers
worldwide (Brenner et al., 2014), and its incidence is constantly
increasing with an estimated 2.4 million cases in 2035, due to
modern diet and lifestyle, along with reduced physical activity.
Current efforts are not sufficient to combat the present epidemic
of colorectal cancer and therefore novel approaches are needed
for effective prevention and treatment including changes in life
style in combination with safer alternative interventions such as
* Corresponding Author
Morana Jaganjac, PhD, Senior Scientist, Toxicology and Multipurpose
Labs, Anti Doping Lab Qatar, Sport City Road, Doha, Qatar.
Phone: +97444132846; Fax: +97444132997
Phytotherapeutics (Weidner et al., 2015). Phytotherapy, the use of
medicinal plants to treat diseases, has been an inevitable part of
ancient human history. Medicinal plants have long been utilized as
alternative treatment sources for cancers, representing more than
sixty percent of anticancer agents used in conventional medicine
(Balunas and Kinghorn, 2005; Saibu et al., 2015).Some of the best
known examples include extracts from Catharanthus roseus G.
Don. (Apocynaceae), Taxus baccata L. (Taxaceae) and
Camptotheca acuminate Decne (Nyssaceae) (Cragg and Newman,
2005; da Rocha et al., 2001). Several herbal extracts and
phytochemicals were shown to exert antitumor effects in colorectal
cancer attributed to the induction of reactive oxygen species (ROS)
production and associated apoptosis of cancer cells as the case of
extracts from Melissa officinalis (Weidner et al., 2015).

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Among phytotherapeutic candidates, Cistanche tubulosa,
an Orobanchaceae parasitic desert plant (Jiang et al., 2009) that is
widely distributed in arid and semi-arid regions of Africa, Asia
and the Mediterranean region, has been shown to have valuable
medicinal properties. Cistanche tubulosa has been extensively
used in traditional medicine, and suggested to have curative effects
in kidney deficiency, morbid leucorrhea, metrorrhagia, female
infertility, and senile constipation (Jiang et al., 2009).In addition to
its traditional medicinal uses, important medicinal properties of
Cistanche Tubulosa have been intensively studied during the last
decade including vasorelaxant (Yoshikawa et al., 2006),
hepatoprotective (Morikawa et al, 2010), anti-hyperglycemic and
hypolipidemic effects (Xiong et al., 2013). Cistanche Tubulosa
was also suggested as a potent enhancer of the immune system,
promoter of bone formation, and an anti-aging and anti-fatigue
agent (Xu et al., 2014). Furthermore, the Cistanche tubulosa
extract has been shown to block amyloid deposition in
Alzheimer’s disease model (Wu et al., 2015). Despite its various
therapeutic uses, the effect of Cistanche Tubulosa as a potential
anticancer agent has not been studied yet.
In the present work we have investigated the
anticancer effect of Cistanche Tubulosa on two primary and two
metastatic colon cancer cell lines and the potential mechanisms
underlying this effect.
MATERIALS AND METHODS
Collection and preparation of plant extract
The samples of Cistanche tubulosa were collected from
desert area in Qatar during 2014 and the authenticity of the plant
was confirmed by herbatologist. Voucher samples are archived in
the Toxicology and Multipurpose Department at ADLQ. The sun-
dried plant samples were grinded with Retsch Knife Mill
Grindomix GM300 into fine powder. Twenty grams of powder
were extracted with 200 mL ultrapure water over night at 37 °C on
a rotary shaker at 200 rpm. The crude Cistanche tubulosa extracts
(CTE) were centrifuged for 30 min at 8000 rpm to pellet non-
soluble compounds, supernatant collected and freeze dried using
Labconco Freezone 6 plus Freeze dryer. The dried extract was
reconstituted in Dulbecco’s Modified Eagle’s Medium (DMEM,
SIGMA, Germany) to a concentration of 20 mg/mL and sterile
filtered through 0.2 micron membrane filter.
Cell lines and cell maintenance
Human colon carcinoma cell lines CaCo2, SW620 and
LoVo were obtained from Cell Lines Service (CLS, Eppelheim,
Germany) while HCT 116 cell line was a kind gift from the
Biological and Environmental Sciences, Department at Qatar
University. CaCo2 and HCT11 were derived from the primary site
of colon carcinoma while SW620 and LoVo derived from
metastatic site. SW620, HCT116 and LoVo cells were cultured
and maintained in DME Medium while CaCo2 cells were
maintained in Eagle's Minimum Essential Medium (EMEM,
SIGMA, Germany). The cells were grown as monolayer cultured
at 37°C in respective medium supplemented with 10% fetal bovine
serum (FBS, SIGMA, Germany) and 1% Penicillin/Streptomycin
(SIGMA, Germany) in a humidified atmosphere containing 5%
carbon dioxide.
Cell viability assay
The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
(MTT) assay was used to evaluate the cytotoxic activity of CTE
similarly as described earlier (Jaganjac et al., 2010). The seeding
density of CaCo2, HCT116, SW620 and LoVo cells cultured in
96-well plates was 104 cells per well. Cells were plated in
respective medium supplemented with 10% FBS 24 hours prior to
treatment. After 24 hours, the medium was removed and cells were
treated with 0, 0.25, 0.5, 1 and 2 mg/mL of CTE for 24, 48 and 72
hours at 37°C in a humidified atmosphere containing 5% CO2.
Upon CTE treatment, the medium was removed and 40 µL of
MTT solution (0.5 mg/mL) added to each well.
After 3 h of incubation, MTT solution was removed, the
formazan product dissolved in dimethylsulfoxide (DMSO,
SIGMA, Germany), and the absorbance measured at 590 nm with
microplate reader (Infinite 200 PRO NanoQuant, Tecan Trading
AG, Switzerland).
Apoptosis assay
Apoptosis in CaCo2, HCT116, SW620 and LoVo cells
was detected using PE Annexin V Apoptosis Detection Kit I with
7-Amino-Actinomycin D (7-AAD) as a vital dye (Becton
Dickinson International, Belgium) according to the manufacturer's
instruction. Briefly, cells were seeded in 24-well plates at a density
of 5×104 cells/well in a respective media supplemented with 10%
FBS for 24 h prior to the addition of CTE (0, 0.5 or 1 mg/mL).
Following 24 CTE incubation, cells were harvested, washed twice
with cold phosphate-buffered saline and stained with
Phycoerythrin (PE) Annexin V and 7-AAD for 15 min at room
temperature in the dark. Stained cells were analyzed within 1 hour
by flow cytometry using FACS. Aria III flow cytometer and
FACSDiva software (Becton Dickinson) at a low flow rate with a
minimum of 104 cells. Treatment of cells for 4 hours with 6 µM
camptothecin (SIGMA) was used as a positive control for the
assay.
Intracellular ROS production
Intracellular ROS production was examined using 2,7-
dichlorodihydrofluorescein diacetate (DCFH-DA, SIGMA,
Germany). DCFH-DA is a nonfluorescent probe, which is oxidized
with intracellular ROS to the fluorescent compound 2,7-
dichlorofluorescein (DCF) (Poljak-Blazi et al., 2011). The DCFH-
DA assay was performed similarly as we have described before
(Cindric et al, 2013; Poljak-Blazi et al., 2011). Briefly, the seeding
density of CaCo2, HCT116, SW620 and LoVo cells cultured in
96-well black plates was 104 cells per well. Cells were plated in
respective medium supplemented with 10% FBS for 24 hours.
Prior to treatment cells were incubated with 10 µM DCFH-DA at
37°C for 30 min in 5% CO2 / 95% air. Cells were then washed and

Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 041
treated with 0, 0.5 and 1 mg/mL of CTE in the medium w/o phenol
red. The intracellular ROS formation was monitored continuously
throughout 25 hours at 37°C and 5% CO2 using microplate reader
with top fluorescence and gas control module (Infinite 200 PRO,
Tecan Trading AG, Switzerland). The fluorescence intensity was
measured with an excitation wavelength of 500 nm and emission
detection at 529 nm. The arbitrary units, relative fluorescence units
(RFU), were based directly on fluorescence intensity.
Mitochondrial superoxide generation
The ability of CTE to induce superoxide generation by
mitochondria was estimated using cell-permeable, mitochondria
targeted MitoSOX Red probe (Life Technologies) and Hoechst
33342 for nuclear staining (Life Technologies). CaCo2, HCT116,
SW620 and LoVo cells were seeded in 96-well plates at a density
104 cells per well in a respective medium supplemented with 10%
FBS for 24 hours. Cells were then loaded with 4 µM of MitoSOX
and 2 µM of Hoechst 33342 for 20 min, excess dye washed and
wells treated with 0, 0.5 and 1 mg/mL of CTE for 24 hours at 37°C
and 5% CO2. The fluorescence intensity was measured with an
excitation wavelength of 510 nm and emission detection at 580 nm
for MitoSOX and an excitation wavelength of 350 nm and
emission detection at 461 nm for Hoechst 33342 using microplate
reader with top fluorescence (Infinite 200 PRO, Tecan Trading
AG, Switzerland)
Statistical analysis
Descriptive statistics were shown as the mean +/− SD.
The significance of differences between groups was assessed using
the Student t-test and Chi-square test. When more than two groups
were compared, we used one sided ANOVA with appropriate post
hoc testing. The SPSS 11.01 for Mircosoft Windows were used.
Differences with P less than 0.05 were considered statistically
significant.
RESULTS
The effect of CTE on proliferation of human colon
cancer cell lines is shown in Figure 1. All CTE concentrations
tested showed a strong inhibitory effect on CaCo2 cell line in a
concentration and time dependent manner (Figure 1A). Seventy
two hours of CTE treatment inhibited the growth of CaCo2 cells
by more than 60% compared to control (p < 0.05 for all
concentrations). Significant impact of CTE treatment on HCT116
cell growth was also detected at all-time points at the two highest
concentrations (1 mg/mL and 2 mg/mL), reaching greater than
70% reduction at latter concentration (Figure 1B, p < 0.05).
Although the two lower CTE concentrations (0.25 and 0.5 mg/mL)
significantly reduced HCT116 growth of cells after 24 hours
(p<0.05), they had no significant effect following 72 hours
treatment compared to control (p>0.05). Time and concentration
dependent inhibition of proliferation with CTE was further
confirmed in LoVo cells by more than 60% at highest
concentration (p < 0.05) (Figure 1C), and all four CTE
concentrations tested reduced the growth of SW620 cells after 48
hours (Figure 1D, p < 0.05 for all). After 72 hours of treatment the
same effect was observed only for the two highest concentrations
(p < 0.05) while 0.25 and 0.5 mg/mL concentrations showed no
significant effect compared to control (p > 0.05). The impact of 0.5
and 1 mg/mL CTE treatment on induction of apoptosis was further
tested in all four cell lines (Figure 2). Increased number of cells in
early apoptosis was detected in HCT116 and LoVo following 24
hours treatment with 0.5 mg/mL (p<0.05, Figure 2B and 2C) and
in all cell lines at 1 mg/mL (p<0.05). Significant increase in
necrotic or late apoptotic cell number was further observed in
CaCo2 and SW620 cell lines (p<0.05, Figure 2A and 2D). Ability
of CTE to induce intracellular ROS production is demonstrated in
Figure 3. Three hours following CTE treatment there was a strong
increase in intracellular ROS production in all cell lines (p<0.05).
The intracellular ROS production increased progressively
throughout the 25 hours treatment in a time and concentration
dependent manner. Furthermore, staining of cells with
mitochondria targeted probe revealed a strong impact of CTE on
mitochondrial superoxide production in a concentration dependent
manner (Figure 4). Highest increase in superoxide production by
mitochondria was observed in HCT116 (69%, Figure 4B) and
LoVo cells (82%, Figure 4C) following 24 hours treatment with 1
mg/mL of CTE.

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Fig. 1: Effect of Cistanche tubulosa on the viability of colon cancer cell lines. Cell viability measured by MTT assay of (A) CaCo2, (B) HCT116, (C) LoVo and
(D) SW620 cells are presented as percentage of control untreated colon cancer cell line. Mean values (±SD) for 5-replicates of representative experiment is
given: (*) significance p<0.05 in comparison to control untreated respective cells.
Fig. 2: Cistanche tubulosa water extract induces apoptosis in human colon cancer cells. Annexin-V-FITC flow cytometry analyses of (A) CaCo2, (B) HCT116,
(C) LoVo and (D) SW620 cells are presented as percentage of control untreated colon cancer cell line. Mean values (±SD) for 3-replicates of representative
experiment is given: (*) significance p<0.05 in comparison to control untreated respective cells.

Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045 043
Fig. 3: Cistanche tubulosa water extract induces intracellular ROS production in a time- and dose-dependent. ROS production measured by DCFH-DA assay in
(A) CaCo2, (B) HCT116, (C) LoVo and (D) SW620 cells is presented as mean RFU values (±SD) for the respective 5-replicates of representative experiment.
(*) Significance p<0.05 in comparison to control untreated colon cancer cells.
Fig. 4: Cistanche tubulosa water extract induces mitochondrial superoxide production in human colon cancer cells. Fluorescence intensity of mitochondria
targeted MitoSOX Red probe in A) CaCo2, (B) HCT116, (C) LoVo and (D) SW620 cells are presented as percentage of control untreated colon cancer cell line.
Mean values (±SD) for 5-replicates of representative experiment is given: (*) significance p<0.05 in comparison to control untreated colon cancer cells.

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Al-Menhali et al/ Journal of Applied Pharmaceutical Science 7 (05); 2017: 039-045
DISCUSSION
Although earlier studies have reported numerous
medicinal properties of Cistanche tubulosa, this is the first report
of its anti proliferative effect in malignant cells. Cistanche
tubulosa bioactive compounds extracted with water, a highly polar
solvent, showed strong anticancer bioactivity. We have previously
compared the efficiency of Cistanche tubulosa solubilized in water
against other solvents such as methanol and ethyl acetate, but
water extracts exhibited the most promising anticancer activities
(data not shown).
We have demonstrated the ability of CTE at 1 mg/mL
and 2 mg/mL to inhibit 60% of the growth of both primary and
metastatic colon cancer cell lines, revealing a potential important
role of Cistanche tubulosa as a colon cancer treatment. Compared
to normal cells, cancer cells are generally characterized by a
disturbance in redox homeostasis and a common strategy of
current anticancer therapies is to increase cellular oxidative stress
(Yang et al, 2013). Although physiologically low levels of ROS
have important role as signaling molecules, the excessive ROS
production can contribute to cancer instability and malignancy
(Liou and Storz, 2010). Paradoxically, this imbalance in cellular
redox homeostasis renders cancer cells more vulnerable to ROS-
induced cell death (Jaganjac et al., 2008; Nogueira and Hay,
2013). The anti-proliferative effect of CTE reported in this study
can be mediated by various extra- and intracellular mechanisms of
known and unknown compounds within the extract, targeting
multiple pathways that play essential roles in apoptosis. In order to
differentiate between different modes of cell death, we
investigated the potential mechanism responsible for the observed
CTE-induced cytotoxicity.
Our data indicate that CTE increases intracellular ROS
production and consequently ROS-induced cell death. Redox state
of the cell also plays a crucial role in regulating apoptosis and
mitochondrial electron transport chain is one of the major sites of
cellular ROS generation (Trachootham et al., 2008). Furthermore,
the intracellular ROS could cause cellular apoptosis via both
mitochondria-dependent and independent pathways (Sinha et al.,
2013). Indeed, our data also indicate that CTE induced
phosphatidylserine externalization, a common effect in apoptosis,
in both primary and metastatic cancer cell lines, suggesting that
the mechanism of CTE induced death is mediated by apoptosis
rather than necrosis. The activation of apoptosis in cancer cells is a
corrective strategy and many anticancer drugs may exert apoptotic
effects in cancer cells.
Compounds or extracts with pro-apoptotic activities in
cancer cells are therefore potentially useful in anticancer drug
research (Wong, 2011). In order to determine whether CTE
induced pro-apoptotic effect is mediated by mitochondria-induced
ROS mechanism, we measured superoxide production using
mitochondria targeted fluorescent probe in CTE treated cells.Our
data clearly shows that CTE stimulates mitochondrial superoxide
production suggesting that Cistanche tubulosa anticancer activity
is at least in part mediated through mitochondria induced ROS
mechanism.
CONCLUSIONS
In conclusion, our data suggests that the water extract of
desert plant Cistanche tubulosa may represent a promising
candidate for anticancer approach in combination with other
conventional therapies for prevention and treatment of colon
cancer. We also demonstrate that the toxicity of the plant extract
against cancer cells is mediated by increased intracellular ROS
production and, at least in part, by mitochondrial-dependent
apoptosis. Further studies are ongoing to isolate and characterize
the individual biologically active constituents responsible for
anticancer activity.
More research is needed to evaluate the potential use of
this extract as an effective chemopreventive agent and to
understand the mechanisms of action on colon cancer cells at the
molecular level. Further preclinical and clinical studies are also
needed to confirm the observed beneficial health effects of
Cistanche tubulosa for cancer prevention.
ACKNOWLEDGEMENTS
Financial support and sponsorship: This study was supported by
the Anti Doping Lab Qatar.
Conflict of Interests: The authors’ declare no conflict of interest.
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How to cite this article:
Al-Menhali AS, Jameela SA, Latiff AA, Elrayess MA, Alsayrafi
M, Jaganjac M. Cistanche tubulosa induces reactive oxygen
species-mediated apoptosis of primary and metastatic human colon
cancer cells. J App Pharm Sci, 2017; 7 (05): 039-045.