Vanadium complex: an appropriate candidate for killing hepatocellular carcinoma cancerous cells

Article abstract of DOI:10.1007/s10534-018-0139-x

Hepatocellular carcinoma (HCC) is a prevalent human malignancy which its drug resistance is increasing world-wide. This project was designed to assess the anti-cancer effects of 4-bromo-2-(((5-chloro-2-hydroxyphenyl) imino) methyl) phenol ([IV(L)] complex

Full text of DOI:10.1007/s10534-018-0139-x

Biometals
https://doi.org/10.1007/s10534-018-0139-x(0123456789(). -, volV
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Vanadium complex: an appropriate candidate for killing
hepatocellular carcinoma cancerous cells
.
Hamid Bakhshi Aliabad Soudeh Khanamani Falahati-pour Hadis Ahmadirad
.
.
.
.
Maryam Mohamadi Mohammad Reza Hajizadeh Mehdi Mahmoodi
Received: 19 March 2018 / Accepted: 4 September 2018
Ó The Author(s) 2018
Abstract Hepatocellular carcinoma (HCC) is a
prevalent human malignancy which its drug resistance
is increasing world-wide. This project was designed to
assess the anti-cancer effects of 4-bromo-2-(((5-
chloro-2-hydroxyphenyl) imino) methyl) phenol
revealed that vanadium m, [IV(L)] complex induce
early apoptosis higher in HepG2 cell line than L929
cells. The rates of necrosis/late apoptosis were also
induced in HepG2 cells more than L929 cells. Based
on the results, vanadium m, [IV(L)] complex might be
considered as a safe new drug for treatment of HCC
with low side effects on control liver cells.
(
[IV(L)] complex) on the HepG2 cell line and also
L929 cells, as normal cells. HepG2 and L929 cells
were cultured in RPMI culture medium and the
survival rates of the cells were determined after 24
and 48 h using MTT assay to find IC50 concentration
of vanadium m, [IV(L)] complex. The early apoptosis
and necrosis/late apoptosis were determined by means
of annexin V/PI apoptosis detection kit. The results
Keywords Vanadium complex Á HepG2 cells Á L929
cells Á Hepatocellular carcinoma Á Cytotoxicity
Abbreviations
HCC
Hepatocellular carcinoma
Vanadium m, [IV(L)] IV complex with 4-bromo-2-
complex
(((5-chloro-2-
H. Bakhshi Aliabad Á H. Ahmadirad Á
hydroxyphenyl) imino)
methyl) phenol (L)
Fetal bovine serum
Ethylene diamine tetra acetic
acid
M. R. Hajizadeh Á M. Mahmoodi
Molecular Medicine Research Center, Research Institute
of Basic Medical Sciences, Rafsanjan University of
Medical Sciences, Rafsanjan, Iran
FBS
EDTA
S. Khanamani Falahati-pour Á M. Mohamadi
Pistachio Safety Research Center, Rafsanjan University of
Medical Sciences, Rafsanjan, Iran
MTT
3-(4,5-Dimethylthiazol-2-
yl)-2,5- diphenyl tetrazolium
bromide
M. R. Hajizadeh Á M. Mahmoodi
Department of Clinical Biochemistry, Faculty of
Medicine, Rafsanjan University of Medical Sciences,
Rafsanjan, Iran
Introduction
M. Mahmoodi (&)
Department of Clinical Biochemistry, Afzalipoor Faculty
of Medicine, Kerman University of Medical Sciences,
Kerman, Iran
It has been demonstrated that cancers are the main
cause of mortality in the human population (Winters
e-mail: mahmoodies@yahoo.com
123

Biometals
et al. 2017; Tervonen et al. 2017). The Liver is a main
organ which can be suffered from cancers which are
induced by several factors such as drugs, viruses and
chronic inflammation (Ho et al. 2016). Several thera-
peutic approaches have been introduced for the treat-
ment of cancers including hepatocellular carcinoma
expression molecules involved in the phosphoinosi-
tide-3-kinase-protein kinase B/Akt (PI3K-PKB/Akt),
p21 activated protein kinases (PAK), death-associated
protein kinase (DAPK), cyclin-dependent kinase
(CDK) 4, 6 and 7, Fas-associated protein with death
domain (FADD), protein 2-alpha (AP2), and c-Jun
N-terminal kinase (JNK) signaling pathways (Leon
et al. 2016) and the several molecules such as B cell
(
HCC) including radiotherapy, chemotherapy and
immunotherapy (Shiba et al. 2017; Obeid et al. 2017).
Chemotherapy is a famous approach which is used for
the treatment of several cancers including HCC (Le
Grazie et al. 2017). However, the current drugs which
are used for chemotherapy are associated with several
side effects which are derived from their effects on the
normal non-cancerous cells (Le Grazie et al. 2017;
Ceylan et al. 2015; Clavagnier 2014; Hosseini et al.
lymphoma-extra (Bcl-x), Caspase
3
(CASP3),
CASP6, CASP7, CASP10 and CASP11 (Leon et al.
2017b). However, the effectiveness of the anti-cancer
metals on HCC and also their side effects on normal
cells need to be explored by further investigations.
Based on the fact that HepG2 is a well-known cell
line for using in HCC related investigations, hence, the
main purpose of this study was to appraise the anti-
cancerous effects of an IV complex with 4-bromo-2-
(((5-chloro-2-hydroxyphenyl) imino) methyl) phenol
(L), which abbreviate to [IV(L)] complex, on the
HepG2 cell line. On the other hand, due to the various
side effects of chemotherapy on the normal cells of the
hosts, another goal of this study was to explore the
effects of the [IV(L)] complex on the survival and
apoptosis of L929, a normal cell line.
2
017; Zainodini et al. 2018). Therefore, investigators
have been trying to find new therapeutic strategies for
cancer treatment with the lowest side effects (Sheikh-
rezaei et al. 2018; Karimabad et al. 2017; Ramezani
et al. 2017; Mohammadizadeh et al. 2018). Based on
the fact that HCC is a prevalent cancer word-wide,
hence, several studies are designed to introduce new
chemotherapy strategies to overcome the disease.
Accordingly, several liver cell lines, including HepG2,
have been introduced by investigators to examine new
drugs for the treatment of HCC (Han et al. 2015). Thus,
this cell line has been used in several studies to examine
the effectiveness of new anti-cancer drugs.
Materials and methods
Material and instrumentations
Cell lines
It has been proposed that vanadium (IV), a metal ion
complex, is a suitable candidate for cancer treatment
(
Nair et al. 2014). It appears that vanadium IV has lower
side effects than platinum metal ions (Leon et al.
017a), hence, recent investigations are focused on the
2
Human cancerous (HepG2) and mouse fibroblast L929
cell lines have been purchased from Pasture Institute,
Tehran, Iran.
IV complexes to find a suitable drug with the lowest
side effects on normal non-cancerous cells. Vanadium
compounds as a new class of non-platinum metallo-
drugs have attracted much attention and large efforts
have been made to discover new molecular targets of
these compounds (Leon et al. 2017a; Le o´ n et al. 2016;
Sciortino et al. 2018; Ebrahimipour et al. 2015; Abbasi
et al. 2017; Hong et al. 2017; Schmidt et al. 2017;
Sheikhshoaie et al. 2016; Ebrahimipour et al. 2016;
Nair et al. 2016; Takjoo et al. 2013; Takjoo et al. 2017;
Heidari et al. 2017; Zabin and Abdelbaset 2016;
Mohamadi et al. 2015).
Cell culture
The HepG2 and L929 cell lines were cultured in the
RPMI 1640 culture medium containing 10% of fetal
bovine serum (FBS), 100 IU/mL penicillin and
100 lg/L streptomycin, and incubated in a 5% CO2
incubator at 37 °C (Memert Company, Germany)
(Bagrezaei et al. 2018). To use the cells for examina-
tion regarding survival, apoptosis and treatment with
the [IV(L)] complex, the growth cells were separated
from culture falcons using trypsin-Ethylene diamine
tetra acetic acid (EDTA) and centrifuged at 1100 rpm
for 5 min. Then, 1 mL fresh RPMI medium was used
Several mechanisms have been proposed for IV
complexes to overcome cancer cells including up-
regulation of free radical reactions which is toxic for
cancer and normal cells (Wang et al. 2010) altered
1
23

Biometals
to make a suspension from the precipitated cells to
determine the survived cells percent via MTT assay
vibration confirming the keto form of the ligand in the
solid state. In solution, however, the enol tautomer
was the prominent form of the ligand. After the loss of
the enolic proton in complexation process, a dianionic
ligand was produced (L2-). The band attributed to
(
Roche CO. Mannheim, Germany) and determination
of inhibiting cell growth by 50% (IC50).
-
1
Preparation of 4-bromo-2-(((5-chloro-2-
hydroxyphenyl)imino)methyl)phenol (H2L)
azomethine moiety was observed at 1627 cm in the
-
1
H2L spectrum and at 1596 cm in the [VOL(bipy)]
spectrum. This shift suggested the coordination of the
azomethine nitrogen to the V(IV) ion. Due to the
coordination, the C–O vibration appeared at
Five milliliter of an ethanolic solution containing
0
.03 g (0.2 mmol) 2-amino-4-chlorophenol was added
-
1
-1
to 5 mL solution of equimolar quantity of 2-hydroxy-5-
bromobenzaldehyde in ethanol while stirring vigor-
ously. After refluxing the mixture for 30 min, a red
precipitate (H2L) was formed. The product was
separated by filtration, washed with cold ethanol, and
dried in a desiccator over anhydrous CaCl2.
1320 cm in the ligand shifted to 1289 cm in the
V(IV) complex confirming the coordination of the
oxygen to the metal central ion. The V = O vibration
-
1
of the vanadyl was observed at 949 cm (Fig. 1).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay
m.p.: 199 °C. Anal. Calcd for C1 H
3
9-
-
BrClNO (326.57 g mol ): C, 47.81; H, 2.87; N,
1
2
4
.29. Found: C, 47.79; H, 2.82; N, 4.32%. FT-IR
The MTT cell proliferation assay was used to deter-
mine IC50 for the [IV(L)] complex. Briefly, HepG2
(
KBr) cm: m(OH) 3445, m(N–H) 3131, m(C = N)
4
1
627, m(C = Cring) 1580, m(C–O) 1320, m(C–Cl)
80, m(C–Br) 585.
and L929 cells were seeded at a density of 1 9 10
6
cells/well and treated with the [IV(L)] complex at
several concentrations for 24 and 48 h. Accordingly,
10 lL MTT (5 mg/mL) (Roche CO. Mannheim,
Germany) was added to each wells and incubated at
37 °C for 1 h After solving of formazan crystal in 200
lL of DMSO, the absorbance of the samples was
measured at 570 nm with background subtraction at
630 nm. The IC50 of the [IV(L)] complex for HepG2
and L929 cell lines were then calculated to use for
analysis of apoptosis by Annexin-V/PI apoptosis
detection kit (Ebioscience, CA, USA). To evaluate
the time-dependent effect, viability of HepG2 and
L929 cells at IC50 value was determined at both 24
and 48 h.
0
Preparation of (2,2 -bipyridine)[4-bromo-2-((5-
chloro-2- hydroxyphenylimino)methyl)phenol]
oxido-vanadium(IV) [VOL(bipy)]
To 6 mL hot methanolic solution of H2L (0.1 mmol,
0
.03 g), an equimolar amount of VOSO Á3H O
4
2
(
0.1 mmol, 0.02 g) was added. A brown solution
was obtained which was refluxed for 1 h. After adding
0
0
.1 mmol (0.02 g) of 2,2 -bipyridine to the mixture,
the reflux was continued for further 3 h. Slow
evaporation of the solvent resulted in suitable single
crystals [VOL(bipy)]. The product was separated,
washed with cold ethanol, and dried in a vacuum
desiccator over CaCl2.
Apoptosis evaluation
3
m.p.: 236 °C. Molar conductivity (1.0 9 10 M,
-1
-2
-1
DMSO): 20.8 X cm mol . Anal. Calcd for C
H BrClN O V (579.72 g mol ): C, 49.72; H, 3.30; N,
3 4
Annexin-V-FITC and propidium iodide for analysis
of apoptosis
24-
-1
19
7.25. Found: C, 49.67; H, 3.28; N, 7.29%. FT-IR (KBr)
cm1: m(C = N) 1596, m(C = Cring) 1511 s, m(C–O)
Apoptosis was determined using a commercial
annexin V-FITC/PI apoptosis detection kit (eBio-
science Co. San Diego, CA, USA). Accordingly, the
seeded HepG2 and L929 cells which were pre-treated
with IC50 concentration of the [IV(L)] complex for 24
and 48 h, were harvested and washed using cold PBS.
The washed cells were incubated with 500 lL binding
buffer which contains 1.25 lL of annexin V-FITC and
1
289 m, m(V = O) 949 s, q(pyring) 905 m, m(C–Cl)
01 m, m(C–Br) 647w.
7
FTIR spectral data
In the IR spectrum of H2L, the band appeared at
-
1
3
131 cm was assigned to the NH…O stretching
123

Biometals
2
Fig. 1 Demonstrates FTIR spectrum of H L (a), FTIR spectrum of [IV(L)] (b)
1
0 lL of propidium iodide (PI) for 45 min. The stained
and un-stained cells were analyzed using Cyflow
Evaluation of morphological changes in the treated
cells
Ò
space flow cytometer (Partec, M u¨ nster, Germany).
Flowmax software (Partec) was used for data analysis.
The treated cells were also evaluated regarding the
alteration in cell morphology, which is a main result of
apoptosis. Accordingly, morphological changes on
1
23

Biometals
Fig. 2 Determination of IC50 in L929 and HepG2 cell lines
after 24 and 48 h culture. a Illustrates the percent of cell
viabilities in L929 and HepG2 cell line at 24 h, b illustrates the
percent of cell viabilities in L929 and HepG2 cell lines at 48 h,
respectively. MTT test revealed that IC50 for L929 cell line at
24 and 48 h were 100 and 69, respectively. IC50 for HepG2 cell
line at 24 h was 100 and at 48 h was 79 lg/mL. Cell viability in
DMSO and control groups were 100%
HepG2 and L929 cells after exposure to [IV(L)]
complex were examined using an inverted microscope
under 9 400 magnification. P value of less than 0.05
was considered statistically significant.
of 3.67 lg/mL (0.012 lL) at 48 h, these values are
large. However, the [IV(L)] complex can be consid-
ered and further studied as a potent anticancer agent.
The analysis of results showed that the [IV(L)]
complex IC50 values for HepG2 cells at 24 and 48 h
were 0.17 lL or 100 lg/mL and 0.14 lL or 79 lg/
mL, respectively. The IC50 value for L929 cells at
24 h was 0.17 lL or 100 lg/mL and at 48 h was 0.
12 lL or 69 lg/mL (Fig. 2). The morphologic
images of L929 and HepG2 cell lines before and after
treatment with [IV(L)] complex are presented in the
Fig. 3.
Statistical analysis
After evaluation of raw data regarding Gaussian
distribution, One Way ANOVA under SPSS software
version 18 was used to analyze the data.
Results
Flowcytometry analysis revealed that early and
necrosis/late apoptosis rates in the L929 control cells
(Untreated) by [IV(L)] complex) were 0.20 and 0.05%
after 24 h and 1.09 and 0.01% after 48 h, respectively.
However, early and necrosis/late apoptosis rates in the
L929 cells which were under treatment of [IV(L)]
complex in IC50 concentration were 48.75 and 1.97%
after 24 h and 36.85 and 0.10% after 48 h,
respectively.
The results demonstrated that although, the IC50 for
both cell lines at 24 h was the same (100 lg/mL or
0
.17 lL), it was higher for HepG2 (79 lg/mL or
.14 lL) than L929 (69 lg/mL or 0.12 lL) at 48 h.
0
Based on the results it seems that L929 cells, as the
normal liver cells, were more sensitive to [IV(L)]
complex than the cancerous cells (HepG2). Compared
with the clinical drug reference cisplatin with the IC50
123

Biometals
Fig. 3 Morphology of L929 and HepG2 cell lines before and
after treatment with [IV(L)] complex. [IV(L)] complex induces
apoptosis in both cell lines. a L929 control before treatment with
IV complex. b L929 after treatment with [IV(L)] complex.
c HepG2 control before treatment with [IV(L)] complex and
d HepG2 after treatment with [IV(L)] complex, respectively
The early apoptosis rate in HepG2 control cells
Discussion
(
without treatment with the [IV(L)] complex) at 24 h
was 0.39%, while its necrosis/late rate was 0.02%.
Early and necrosis/late apoptosis rates in HepG2
control cells at 48 h were 1.24% and 0.60, respec-
tively. Additionally, early (44.96%) and necrosis/late
HCC is a prevalent cancer, especially in the viral
hepatitis endemic region (Winters et al. 2017; Tervo-
nen et al. 2017). It has been reported that HCC showed
drug resistance in some cases (Galun et al. 2017; Dong
et al. 2017; Guo et al. 2014) and, hence, investigators
are studying new drug candidate to treat the malig-
nancy. However, side effects of the chemical drugs are
the main limitations of using these drugs (Yan et al.
2014).
(
4.35%) apoptosis rates in HepG2 treated cells at 24 h
were significantly increased by the [IV(L)] complex.
Evaluation of early and necrosis/late apoptosis rates in
HepG2 treated cells after 48 h also demonstrated that
the rates of early (51.69%) and necrosis/late (28.36%)
apoptosis rates were increased significantly (Fig. 4
and Table 1).
The results demonstrated that although, the IC50
for both cell lines at 24 h was the same (100 lg/mL or
0
.17 lL), it was higher for HepG2 (79 lg/mL or
.14 lL) than L929 (69 lg/mL or 0.12 lL) at 48 h.
0
1
23

Biometals
Fig. 4 Flow cytometry analysis of HepG2 and L929 cells
treated with [IV(L)] complex. Un-treated [IV(L)] complex cells
for 48 h (f) stained with Annexin V-fluorescein isothiocyanate
(FITC) and propidium iodide (PI). Subsequently, apoptotic and
necrotic cells were quantified by flow cytometry. The different
subpopulations were defined as Q1 annexin V-negative but PI-
positive, i.e. necrotic cells; Q2 annexin V/PI double positive, i.e.
late apoptotic cells; Q3 annexin V/PI double negative, i.e.
normal live cells; Q4 annexin V-positive but PI negative, i.e.
apoptotic cells
(
[
a), HepG2 cells treated at IC50 concentration (100 lg/mL) of
IV(L)] complex for 24 h (b), HepG2 treated at IC50
concentration (79 lg/mL) of [IV(L)] complex for 48 h (c), un-
treated L929 cells (d), L929 treated at IC50 concentration
(
100 lg/mL) of [IV(L)] complex for 24 h (e), and L929 cells
treated at IC50 concentration (69 lg/mL) of [IV(L)] complex
Table 1 Apoptosis percentage at IC50 concentration of [IV(L)] complex (lm/mL)
Apoptosis rates
HepG2
4 h (100 lg/mL) (%)
L929
2
48 h (79 lg/mL) (%)
24 h (100 lg/mL) (%)
48 h (69 lg/mL) (%)
Normal live cells
Early apoptosis cells
Late apoptosis cells
Necrosis cells
47.21
4.35
43.42
2.55
47.12
1.97
55.57
0.10
44.96
3.48
51.69
2.34
48.75
2.16
36.85
7.47
Based on the results it seems that L929 cells, as the
normal liver cells, were more sensitive to [IV(L)]
complex than the cancerous cells (HepG2). Compared
with the clinical drug reference cisplatin with the IC50
of 3.67 lg/mL (0.012 lL) at 48 h, these values are
large. However, the [IV(L)] complex can be
123

Biometals
considered and further studied as a potent anticancer
agent (Leon et al. 2017a; Le o´ n et al. 2016; Sciortino
et al. 2018; Ebrahimipour et al. 2015; Abbasi et al.
vanadium induces apoptosis in SiHa and HeLa
cancerous cell lines via up-regulation of p53, the
well-known pro-apoptotic protein (Nair et al. 2014).
Wu and colleagues also revealed that Sodium ortho-
vanadate, another component of Vanadium, sup-
presses human HCC cells in both in vitro and
orthotopic in vivo model via modulation of prolifer-
ation, cell cycle, apoptosis and autophagy (Wu et al.
2014). The cancer prevention roles played by vana-
dium were also demonstrated by Mandair et al. (2014)
and Bishayee et al. (1997). Interestingly, Kowalski
et al. reported that vanadium complexes not only
induces apoptosis in human pancreatic ductal adeno-
carcinoma cell line, but also leads to induction of
necroptosis in the cell lines (Kowalski et al. 2017). Our
results also showed that the [IV(L)] complex can
induce apoptosis in HepG2 cell lines more than L929
cells, and also induces necrosis/late apoptosis in
HepG2 cells higher than normal liver cells which
can be associated with anti-cancer inflammation.
Therefore, our results confirmed the apoptotic and
necrotic roles played by vanadium complexes against
the cancers. Additionally, based on the results, L929
cells were more sensitive than HepG2 to [IV(L)]
complex and have lower IC50 at 48 h incubation.
Based on the investigation by Wang et al. (2010)
which demonstrated that using antioxidants has syn-
ergistic effects with vanadium to kill cancer cells and
also reduce their toxicities on human normal cells, it
may propose that using [IV(L)] complex in association
with antioxidants materials can reduce L929
sensitivities.
2
017; Hong et al. 2017; Schmidt et al. 2017;
Sheikhshoaie et al. 2016; Ebrahimipour et al. 2016;
Nair et al. 2016; Takjoo et al. 2013; Takjoo et al. 2017;
Heidari et al. 2017; Zabin and Abdelbaset 2016;
Mohamadi et al. 2015).
In order to conclude the efficacy of [IV(L)]
complex to be considered as a potential candidate
for cancer therapy need to have a look to the results
obtained from apoptosis and necrosis results. Accord-
ingly, our results showed that the [IV(L)] complex
induces early and necrosis/late apoptosis rates in both
cancer and normal cell lines. However, the rate of
apoptosis, especially early apoptosis, was significantly
higher in the HepG2 cells after 48 h incubation with
[
IV(L)] complex when compared to early apoptosis in
the L929, the normal cell line, cells. Due to the fact
that induction of early apoptosis is an important key
factor for anti-cancer components, hence, based on the
results, it appears that [IV(L)] complex can be
considered as a potential drug to induce apoptosis in
liver tumor cells with lower side effects on normal
cells. Additionally, the early apoptosis was higher
especially in the HepG2 cell lines when it was
incubated with [IV(L)] complex at 48 h. It confirms
that more exposure to [IV(L)] complex can induce
apoptosis in the cancerous cell lines. Interestingly, the
results also showed that necrosis/late apoptosis were
also higher in HepG2 cells than L929 cells and in
another word, just 0.1% of the L929 cell line suffered
from necrosis/late apoptosis (it was 28.36% for
HepG2 cells). Based on the fact that necrosis is an
inflammatory condition which leads to infiltration of
immune cells to the necrotic tissue and consequently
leads to immune-related tissue injuries, it may be
concluded that [IV(L)] complex probably can be
considered as a new HCC treatment strategy because
on induction of inflammation in the HCC tissue
without injuries to normal cell. To the best of our
knowledge, this project is the first investigation on the
biological activity of [IV(L)] complex as a novel anti-
cancer agent. However, to confirm the anti-cancer
effects of the complex, further investigations espe-
cially in the in vivo condition need to be performed to
prove the anti-cancer roles of [IV(L)] complex and
also its safety. In parallel with our results, Nair et al.
reported that nicotinoyl hydrazones component of
Collectively, our results may propose that [IV(L)]
complex can be considered as a new strategy for
treatment of HCC which need to be evaluated in the
in vivo condition. Based on the Wang et al. study, it
may also be hypothesized that [IV(L)] complex may
be modified by co-administration of antioxidant to be
safer than its administration alone.
Acknowledgements This article is an excerpt from Hamid
Bakhshialiabad’s PhD’s thesis in Molecular Medicine Research
Center, Rafsanjan University of Medical Sciences (RUMS),
Rafsanjan, Iran. Authors have a chance to thanks to Molecular
Medicine Research Center staffs, Rafsanjan University of
Medical Sciences for their warmly corporations.
Funding This study was supported by the RUMS by the Grant
Number
(1396.3.2-449)
and
ethical
code:
IR.RUMS.REC.1396.50 from the Rafsanjan University of
Medical Sciences, Iran.
1
23

Biometals
Compliance with ethical standards
Guo YL, Kong QS, Liu HS, Tan WB (2014) Drug resistance
effects of ribosomal protein L24 overexpression in hepa-
tocellular carcinoma HepG2 cells. Asian Pac J Cancer Prev
Conflict of interest The authors report no conflict of interest.
1
5:9853–9857
Ethical approval This article does not contain any studies
with human participants or animals performed by any of the
authors.
Han W, Hou G, Liu L (2015) Polyphyllin I (PPI) increased the
sensitivity of hepatocellular carcinoma HepG2 cells to
chemotherapy. Int J Clin Exp Med 8:20664–20669
Heidari F, Fatemi SJA, Ebrahimipour SY, Ebrahimnejad H,
Castro J, Du sˇ ek M, Eigner V (2017) Six-coordinate oxo-
vanadium (V) dimer complex with methoxy bridging:
Synthesis, crystal structure, biological activity and
molecular docking. Inorg Chem Commun 76:1–4
Informed consent This article does not contain any studies
with human participants.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unre-
stricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative Com-
mons license, and indicate if changes were made.
Ho DW, Lo RC, Chan LK, Ng IO (2016) Molecular pathogen-
esis of hepatocellular carcinoma. Liver Cancer 5:290–302
Hong X-L, Zeng M-H, Liu L-J, Ye X-L, Yi D-S (2017) Syn-
thesis, characterization and in vitro antitumor behavior of a
0
0
0
vanadium (V) complex with 4 -(3-methoxyphenyl)-2, 2 :6
0
0
2 -terpyridine. J Coord Chem 70:1438–1450
Hosseini FS, Falahati-Pour SK, Hajizadeh MR, Khoshdel A,
Mirzaei MR, Ahmadirad H, Behroozi R, Jafari N, Mah-
moodi M (2017) Persian shallot, Allium hirtifolium Boiss,
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