New 13-pyridinealkyl berberine analogues intercalate to DNA and induce apoptosis in HepG2 and MCF-7 cells through ROS mediated p53 dependent pathway: Biophysical, biochemical and molecular modeling studies

Article abstract of DOI:10.1039/c5ra17214d

A new series of 13-pyridinealkyl berberine analogues was synthesized and their DNA binding efficacy studied by employing spectroscopic, calorimetric and molecular modeling techniques. Analogues with more than one CH₂ group showed better intercalative binding than berberine. The analogue with one CH₂ group bound DNA weaker than berberine. The binding of the analogue with single CH₂ group was entropy driven, while those with more than one CH₂ group was favoured by both entropy and enthalpy changes. Higher salt concentration and temperature destabilized the binding. A larger contribution from non-polyelectrolytic forces to the Gibbs energy and the involvement of strong hydrophobic interactions were inferred. Molecular modeling pin pointed the specific binding site and the non-covalent interactions in the association. The best DNA binding analogue (BER5) inhibited the growth of hepatocellular and breast carcinoma most efficiently. It induced apoptosis in HepG2 and MCF-7 cells with externalization of phosphatidylserine and reactive oxygen species generation with accumulation of cells in the G0/G1 phase. Furthermore, up regulation of p53 and p21 indicated the role of p53 in BER5 mediated apoptosis. The results suggested that 13-pyridinealkyl berberine analogues intercalated to DNA much stronger than berberine, the chain length of the linker plays an important role for the binding, and they induced ROS mediated apoptosis in HepG2 and MCF-7 cells by p53 modulation.

Full text of DOI:10.1039/c5ra17214d

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New 13-pyridinealkyl berberine analogues intercalate to DNA and
induce apoptosis in HepG2 and MCF-7 cells through ROS mediated P53
dependent pathway: biophysical, biochemical and molecular modeling
†
studies
a
b
c
c
Sabyasachi Chatterjee, Sumana Mallick, Franco Buzzetti, Gaetano Fiorillo, Tanjia
c
c
b
a*
Monir Syeda, Paolo Lombardi, Krishna Das Saha and Gopinatha Suresh Kumar
aBiophysical Chemistry Laboratory, Organic and Medicinal Chemistry Division, CSIR-
Indian institute of Chemical Biology, Kolkata 700 032, India
bCancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of
Chemical Biology, Kolkata 700 032, India
cNaxospharma srl, Via G. Di Vittorio, 70, 20026 Novate Milanese (MI), Italy
Address for Correspondence
Dr. G. Suresh Kumar, Ph.D
Senior Principal Scientist, Biophysical Chemistry Laboratory
CSIR-Indian Institute of Chemical Biology
4, Raja S.C. Mullick Road, Kolkata 700032, India
Phone: +91 33 2472 4049, Fax : +91 33 2472 3967
Email: gskumar@iicb.res.in
†
Electronic supplementary information (ESI) available, see DOI:
1

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Abstract
A new series of 13-pyridinealkyl berberine analogues were synthesized and their DNA
binding efficacy studied by employing spectroscopic, calorimetric and molecular
modeling techniques. Analogues with more than one CH
intercalative binding than berberine. The analog with one CH
weaker than berberine. The binding of the analog with single CH
driven, while those with more than one CH
2
group showed better
2
group bound DNA
group was entropy
2
2
group was favored by both entropy and
enthalpy changes. Higher salt concentration and temperature destabilized the binding.
A larger contribution from non-polyelectrolytic forces to the Gibbs energy and the
involvement of strong hydrophobic interactions were inferred. Molecular modeling pin
pointed the specific binding site and the non-covalent interactions in the association.
The best DNA binding analogue inhibited the growth of hepatocellular and breast
carcinoma most efficiently. It induced apoptosis in HepG2 and MCF-7 cells with
externalization of phosphatidylserine and reactive oxygen species generation with
accumulation of cells in the G0/G1 phase. Furthermore, up regulation of p53 and p21
indicated the role of p53 in BER5 mediated apoptosis. The results suggested that 13-
pyridinealkyl berberine analogues intercalated to DNA much stronger than berberine,
the chain length of the linker plays an important role for the binding, and they induced
ROS mediated apoptosis in HepG2 and MCF-7 cells by p53 modulation.
2

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Introduction
In cellular system, DNA is frequently targeted by low molecular weight ligands. DNA
targeted therapeutic agents can interfere with numerous biological functions, the most
important being the transcription and replication of DNA. Such interference can
effectively retard or prevent cell division leading to chemotherapeutic action. A large
spectrum of such DNA targeting drugs have been studied since the discovery of the
DNA structure. Some like doxorubicin, dactinomycin, quarfloxin, mitoxantrone,
mitomycin C, cisplatin were clinically employed, while many others are under different
stages of investigation and clinical trial.
Berberine (Scheme 1a), a natural isoquinoline plant alkaloid of the structural class
protoberberines, has many remarkable biological properties, the most potent albeit less
investigated being its anticancer activity. A number of interesting studies, both in vitro
and in vivo, on the antineoplastic activity of berberine have been reported in the
literature that provide potential leads to promising futuristic clinical application for this
alkaloid.1-6 Nucleic acids are believed to be cellular targets of berberine in manifesting
its anticancer activity and consequently many structural studies in this direction have
been performed. An oligonucleotide-berberine complex has been recently crystallized
7
and the structure solved. The DNA binding studies have also been reviewed
extensively in many recent articles.8,9 Berberine has the unique distinction of exhibiting
remarkable adenine-thymine base pair specificity unlike most other anticancer drugs
1
0
that are specific to guanine-cytosine sequences. But the critical limitations of berberine
to be employed as a pharmaceutical preparation are inter alia its poor solubility, low
3

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bioavailability and moderate nucleic acid binding affinity. To circumvent these
problems attempts have been made to form inclusion complexes, nanoformulations,
etc., that have met with only limited success.11-13 It has thus become apparent that more
effective derivatives with enhanced DNA and or RNA binding efficacy are essential for
harnessing the anticancer activity of berberine. Recently a number of berberine (BER)
analogues with aromatic moieties incorporated at the 13-position were reported to be
more effective than berberine in exerting anticancer activity delaying the development
of tumors.5,14-15
In this study, a series of novel 13-pyridinealkyl berberine analogues (Scheme 1b) were
synthesized and their DNA binding activity investigated by various biophysical
experiments and molecular modeling studies. These analogues are named BER1, BER2,
BER3, BER4 and BER5 based on the number of methylene groups in the linker chain.
We observed the most efficient binding with BER5 bearing the highest number of CH
2
groups in the series. Furthermore, these 13-pyridinealkyl berberine analogues induced
apoptosis in HepG2 and MCF-7 human tumour cell lines. Reactive oxygen species
1
6
(
ROS) play pivotal roles in DNA damage and p53 plays a role as downstream effecter
17
of ROS. The function of redox homeostasis in p53-regulated processes is a topic of
great interest. Taken together, this work provides new fundamental knowledge and
molecular mechanisms for the anticancer action of berberine analogues that may help
further development as antineoplastic therapeutics.5,6,18,19
4

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Results and discussion
Characterization of specific alkaloid-DNA association by spectroscopic analysis
These 13-pyridinealkyl berberine analogues possess an extra anchoring site. The
binding of these analogues was initially probed by absorption spectroscopy titration
methodology. The 13-pyridinealkyl analogues, like berberine, have characteristic
absorption spectra in the region of 300−550 nm. The major peak maximum of the
analogues varied around 340 nm and the spectral nature and features were similar. In
this region DNA has negligible absorbance.
In the absorption spectral titration, hypochromic and bathochromic effects were
observed with the occurrence of isosbestic points for all the analogues on titration with
DNA. The representative absorbance titration profile of BER5 with Calf thymus (CT)
DNA is depicted in Fig. 1A. The titration profiles of other analogues are presented in
†
Fig. S1 (ESI ). The changes observed in the absorption spectra of BER5 are characteristic
features of DNA intercalating ligands, revealing that the isoquinoline chromophore is
stacked between the DNA base pairs in an intercalated posture. Equilibrium binding is
confirmed from the presence of isosbestic points involving two state systems
comprising of only free and bound alkaloid analogues. The alkaloid molecule along
with the substituent moiety enters inside the DNA helix in such way that it is unable to
form H-bonds with the solvent water molecules. The Scatchard plots of binding and
†
2
0
subsequent McGhee-von Hippel analysis of the absorption data (Fig. S2, ESI ) revealed
that the BER shows cooperative binding manifested by negative slope at low r values.
5

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†
These are described in details in the ESI . On the other hand, the analogues present a
non-cooperative binding mode with positive slope at low r values. So, the substitution
of a pyridinealkyl group at the 13-position dramatically changes the nature of the
binding from a non-cooperative mode to cooperative mode. This feature of these
analogues is similar to that observed for 9 and 13-substituted analogues reported
previously.18,19,21 The equilibrium binding affinity values and the number of excluded
sites i.e., the number of base pairs excluded by the binding of an alkaloid as per
neighbor exclusion model, on the binding of these analogues are depicted in Table S1
†
(
ESI ). The highest binding constant was observed for BER5 with five CH
2
groups in the
linker and the affinity increased with increasing chain length except for BER1 where
affinity was lower than BER. The number of excluded sites decreased with increasing
chain length and was 6.6, 3.4, 3.2, 3.2 and 3.2, respectively, for BER1-5.
Berberine presents a weak intrinsic fluorescence property. The singlet excited state of
the alkaloid returns to the ground state due to its intersystem crossing via
2
2
nonradioactive mechanism. The 13-pyridinealkyl berberine analogues exhibited more
intense fluorescence than BER. The fluorescence intensity enhanced remarkably on
binding with DNA. A representative spectral titration in the case of BER5 is presented
in Fig. 1B and the titration profiles of analogues BER1-BER4 are presented in the Fig. S3
†
(
ESI ). Change in the hydrophobic environment of the chromophore of the ligand due to
transfer from a high polarity solvent environment to low polarity DNA interior when
intercalated and consequent electronic interactions appear to cause this enhanced
6

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fluorescence intensity. Furthermore, in the solution state rotational movement of the
free alkaloid analogue molecules favor a radiation less deactivation of the excited states.
But when bound to DNA in the intercalative mode the rotation is hindered and the
deactivation through fluorescence emission is favoured, and a significant increase in the
fluorescence intensity occurs.
Conformational changes in DNA measured by circular dichroism spectroscopy
The right handed helical conformation of B-form DNA showed a characteristic CD
spectrum with a positive band in the 275−280 nm region and a negative band around
2
48 nm (Fig. 2A). The long wavelength band arises due to the base stacking interactions
2
3
and the 248 nm band is due to the helicity. The alkaloid analogues have no chiral
center and hence are CD inactive. In an alkaloid-DNA titration study, the molar
ellipticity value of the positive band increased with a small red shift of the wavelength
maximum while the negative band changed only marginally. These features were
similar in all the cases. The maximum change was observed in the case of BER5 as
shown in the Fig. 2A. From the nature of the data it may be divulged that the alkaloid
analogue molecules bind to DNA helix leading to conformational changes within in the
B-form helical organization. It was also observed that a weak induced circular
dichroism band appeared in the 340 nm region as shown in the Fig. 2B which is near the
absorption maxima of the analogues. The result revealed that the alkaloid analogues
bind in between the base pairs of the DNA helix by intercalation and due to this the
helical chirality of DNA has changed. The induced CD spectrum arises from the strong
7

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interaction of the electric transition moments of the analogues and the transition
moments of the chirally arranged DNA bases/base pairs.
Thermal stabilization of the DNA helix by the alkaloids
Optical thermal melting experiments were carried out to understand the stabilization of
the DNA helix on binding of the berberine analogues. The melting profile of DNA,
monitored at the absorbance maximum (260 nm), revealed a cooperative hyperchromic
o
transition of ~40% with a melting temperature (T
m
) around 65 C under the conditions
o
employed here. Berberine stabilized DNA by about 7 C. The binding of BER5 induced
o
o
the highest stabilization and the melting temperature was around 85 C (∆T
m
= 20 C).
o
With the exception of BER1 (∆T
m
= 5 C) all the analogues stabilized DNA more than
BER. The results are presented in Table S2 and the corresponding melting profiles are
†
shown in Fig. S4 (ESI ).
The DNA stabilization was also confirmed by differential scanning calorimetry (DSC)
experiments. The endothermic DNA peak was shifted to higher temperatures due to the
binding of the alkaloid analogues. The ∆T values were 8.00, 4.19, 13.33, 16.75, 17.94
m
o
and 19.57 C, respectively, for BER and BER1 to 5. These values are close to those
†
obtained from optical melting experiments. The results are depicted in Table S2 (ESI ).
Elucidation of the mode of binding of the alkaloid analogues by fluorescence
quenching and viscosity methods
BER is known to bind to DNA by an intercalative mode.7,9,10 But to understand the
mode of binding of the analogues we applied the fluorescence quenching technique and
viscosity measurements. Fluorescence quenching using ferrocyanide ions, an anionic
8

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quencher, has been effectively used to distinguish between intercalation and groove-
1
8
24-26
binding modes of berberine analogues and other small molecules
The Stern−Volmer plots for fluorescence quenching of the BER and its analogues-DNA
†
complexes by ferrocyanide ions are presented in Fig. S5 (ESI ). The results indicated that
free analog molecules were quenched efficiently by the ferrocyanide ions. More
fluorescence quenching was observed in the case of BER1 than BER and the effect
gradually decreased for bound BER2−5, indicating that the bound analogues BER2−5
are located in a relatively more protected environment inside the DNA helix compared
to those unbound. The Stern-Volmer quenching constants (Ksv) calculated were in the
−1
range 215−230 M for the unbound BER and analogues, and 95, 170, 80, 71, 39 and 33
−1
M , respectively, for bound BER and BER1−5. The trend in the values revealed that the
Ksv of BER1 was higher than that of BER and for the others it varied as BER>BER2>
BER3> BER4>BER5. Based on this data it can be inferred that BER1 is a weaker
intercalator than BER, while BER2-BER5 are better intercalators than BER. The pyridyl
group of BER1 and the isoquinoline chromophore are separated by a single (CH ) group
2
and therefore the side chain linker appears to be too close to the isoquinoline
chromophore leading to inhibition of the intercalation BER1. With gradual increase of
the number of CH groups in the linker chain, intercalation capability was found to
2
increase and it became maximum for BER5. This was also clearly evident from the
viscosity data, where the helix length enhancement due to intercalation was maximum
with BER5. A plot of relative increase in length of rod like DNA (L/Lo) versus r, where
r is the number of berberine analog molecules bound per mol of the nucleotide, is
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†
presented in Fig. S6 (ESI ). The slope of the plots (β values) for BER and BER1-BER5
binding to DNA was found to be 0.66, 0.56, 0.70, 0.76, 0.83 and 0.90, respectively. Taken
together, these results confirmed the intercalation of these analogues to DNA and also
suggested a better intercalation geometry for BER2-5 compared to BER and provided
evidence that the linker chain with more than one CH group decisively contributes to
2
the stronger intercalative binding of the isoquinoline moiety.
Thermodynamics of berberine analogues-DNA association
The thermodynamic profile of the interaction, that may provide key insights into the
nature of the molecular forces that drive the complexation, was obtained from
isothermal titration calorimetry (ITC) studies. Figure 3 A, B shows the representative
ITC profiles of BER1 and BER5-DNA interaction. For BER5 the binding was exothermic
in nature (Fig. 3A) but for BER1 (Fig. 3B) it showed endothermic heats. The ITC profiles
in all the other cases were exothermic in nature. The presence of only one binding event
in the thermogram in each case enabled the fitting of the isotherms to ‘one set of
binding sites’ model yielding the best fit curve providing the equilibrium association
o
constant (K), the stoichiometry (N) and the change in enthalpy (∆
r
H ). The entropy
So) and the standard change in molar Gibbs energy (∆
G ) were
o
contribution (T∆
r
r
1
8
calculated as described previously. The data obtained from the ITC experiments are
summarized in the Table 1. A comparative chart depicting the variation of the K values
of the analogues are shown in Fig. 3C. In all the cases except BER1 the binding was
favoured by negative enthalpy and positive entropy changes. In the case of BER1, the
1
0

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binding had an unfavorable enthalpy term. The small positive enthalpy for the binding
was unfavourable to the Gibbs energy. The large entropy contribution in the binding of
Table 1. ITC derived thermodynamic parameters for the binding of berberine analogs
with DNAa
G0)
(∆
H0)
(T∆
S )
0
Alkaloid
analogue
Binding constant
Stoichiometry
(N)
(∆
r
r
r
5
-1
(
K) x10 M
kcal/mol
kcal/mol
kcal/mol
3.92
BER
1.09 ± 0.07
0.46 ± 0.08
2.14 ±0.05
3.27 ± 0.33
6.45 ± 0.08
9.96 ± 0.05
0.482±0.03
0.161±0.08
0.471 ±0.02
0.361±0.07
0.356±0.02
0.352±0.05
-6.75±0.53 -2.834 ± 0.13
BER1
BER2
BER3
BER4
BER5
-6.25± 0.98
-7.07± 0.96
-7.39± 0.90
7.78± 0.61
-8.04± 0.77
0.89 ± 0.18
-1.80 ± 0.06
-1.42 ± 0.09
-1.190 ± 0.06
7.14
5.27
5.97
6.59
-1.04 ± 0.09
7.00
a
o
The ITC experiments were conducted at a pressure of 101.10 kPa at 20 C. K, the
o
binding constant, N, the binding stoichiometry and ∆
r
H , the standard molar
enthalpy change were evaluated from ITC profiles fitting to Origin 7.0 software using
o
the 'one set of binding sites' model. The values of ∆
r
G , standard molar Gibbs energy
change and T∆
r
So, the standard molar entropy contribution were determined using
o
H -T∆
So = -RTlnK. R is the universal gas constant (1.98722 cal
o
the equations ∆
r
G =∆
r
r
K-1 . mol-1.
1
1

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BER1 compared to BER and other analogues can interpreted in terms of release of
bound water and condensed sodium ions from the DNA grooves. This is because the
pyridinealkyl moiety in BER1 appears to be very close to the isoquinoline chromophore
of berberine leading to steric effects preventing BER1 from intercalating strongly
between the DNA base pairs. This is also corroborated from the lower binding affinity
of BER1 compared to all other analogues and even BER. A similar phenomenon was
also reported previously for the DNA binding of 13-phenylalkyl berberine analogues.18
Ionic strength and temperature dependence of the binding
As the berberine analogues are cationic in nature it is expected that ionic interactions
might play a leading role in the complexation process. It was observed that the ionic
strength of the medium had significant influence on the binding of analogues in many
previously reported studies.18,27 To check on the nature of binding forces involved, the
effect of ionic strength on the binding was investigated by ITC experiments in
conjunction with van’t Hoff analysis in the case of the strongest binding analogue, i.e.,
BER5 as a representative case. The thermodynamic parameters of the binding at three
+
+
[
Na ] concentrations investigated, viz. 10, 30 and 50 mM [Na ], were evaluated and the
†
+
results are collated in Table S3 (ESI ). Increased [Na ] concentration results in reduced
electrostatic repulsion between the negatively charged phosphate groups of adjacent
nucleotides, and consequently the binding of BER5 to DNA was hindered, and the
1
2

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equilibrium constant of the complexation decreased; at 30 mM, the binding affinity was
5
-1
+
4
.6×10 M which was about half of that at 10 mM [Na ].
+
A relation linking the equilibrium constant with the Na ion concentration based on
polyelectrolytic theories of Manning’s counter ions condensation model28 has been
derived as follows
∂
log K
(∂
)T , P = −zψ
+
log[Na ]
In this relation, Z is the apparent charge on the berberine analog and ψ is the fraction of
sodium ions bound per phosphate group of the DNA. The number of ions released on
the binding of a single BER5 molecule to DNA was calculated to be - 0.70 from the slope
+
+
of the plot of log K versus log[Na ]. From the dependence of K on [Na ], the observed
Gibbs energy was partitioned between the polyelectrolytic (∆ pe) and non-
r
G
polyelectrolytic (∆
r
t
G ) components using the relationship
0
pe
+
∆
rG = Zψ RTln([Na ])
where Zψ is the slope of the van’t Hoff plot. The values of ∆
salt conditions are depicted in Table S3. It can be observed that the magnitude of ∆
was much lower than that of ∆ at all the salt conditions studied and the latter
Go
r
Gope and ∆
r
Go
t
at the three
Gope
r
r
t
remained more or less constant. So it can be concluded that non-polyelectrolytic forces
dominated the binding interaction here. Similar results were reported for phenylalkyl
substituted berberine analogues also.18
1
3

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Valuable insights into the type and magnitude of forces involved in the binding process
o
was also obtained from the heat capacity (∆
r
Cp ) data derived from the temperature
o
dependant variation of ∆
r
H . Titration calorimetry experiments were performed at
o
three different temperatures, viz. 10, 20 and 30 C, respectively, for the binding of the
analogue BER5. The thermodynamic parameters derived from these experiments are
†
o
presented in Table S4 (ESI ). A plot of ∆
r
H versus T is presented in Fig. 3D. From the
o
slope of the plot the value ∆
r
Cp was determined to be - 25.97 cal/mol K for BER5-DNA
2
9
complexation. For berberine a value of -110 cal/mol K was reported earlier. A negative
heat capacity term is proposed to arise due to the removal of large amount of nonpolar
surface from aqueous exposure upon complex formation resulting in dominant
hydrophobic effects in the interaction and has been displayed by a number of small
molecule binding to DNA and RNA. Deriving strength from these results it may be
proposed that there is a strong hydrophobic element in the interaction of these
berberine analogues with DNA.
Base pair specificity of BER5 from ITC experiments
Berberine is one of the few DNA intercalating molecules known to exhibit remarkable
AT base pair specificity.9,10 To evaluate the binding specificity of the analogues we
studied the binding of BER5, as a representative case, to poly(dA).poly(dT) and
poly(dG).poly(dC). From ITC results the equilibrium constant for the binding to
6
-1
poly(dA).poly(dT) and poly(dG).poly(dC were evaluated to be 2.26 x 10 M and 4.96 x
5
-1
1
0 M , respectively. It may be noted that the AT polynucleotide binding affinity was
about 22 times higher than that to the GC polynucleotide. So it can be ascertained
1
4

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unequivocally that the AT base pair specificity of binding of berberine is faithfully
reproduced in these analogues also.
Elucidation of the binding site of BER5 by molecular docking
Molecular docking studies provide some insight into the interaction between the drug
and DNA, and the results may be correlated with the experimental data. Here we
chosen two oligo nucleotides of different GC content of PDB identifier 258D (A) and
1JH1 (B). The BER5 molecule has two parts, one isoquinoline part (same as berberine)
and an additional spacer arm of alkylpyridine part. In both cases the docking studies
showed that the isoquinoline part is intercalated between the two base pairs of DNA
and the additional alkylpyridine part formed H-bonding interactions with the base of
the DNA molecule as shown in the Fig. 4. The distance between the H-bonding of N-
atom of pyridine (BER5) and the C
.3 Å for N-atom of pyridine and the C
binding energy values from theoretical studies due to binding with the oligomer were -
.59 and -7.90 kcal/mol, respectively. The theoretical studies also supported our ITC
6
-NH
2
group of the adenine (DNA) is 2 Å for A and
2
2
-NH group of the guanine. The calculated
2
8
data of base pair specificity of the BER5 molecule.
Apoptotic effect of BER5 in HepG2 and MCF-7 human tumour cells
To evaluate the efficacy of the BER and its analogues against cancer, the compounds
were tested for their cytotoxic effect against hepatocellular carcinoma (HepG2) and
mammary (MCF-7) human carcinoma cell lines using the MTT assay. After 24 h
treatment, it was found that among the five compounds, BER5 showed a higher degree
of concentration-dependent increase in growth inhibition in HepG2 and MCF-7 cell
1
5

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lines, having GI50 values of 15.7 and 33.2 µM, respectively (Fig. 5A and 5B). However, it
exhibited minimal effect on Human embryonic kidney 293 (HEK-293) cell lines even at a
concentration of 40 µM (Fig. 5C). Treatment with BER5 induced phosphatidylserine (PS)
externalization in HepG2 and MCF-7 cells after 24 h treatment which is a hallmark of
apoptosis. After 24h treatment the percentage of apoptotic cells was 28.1% and 15.6% as
compared to 5.3% and 2.0% in the vehicle treated cells in HepG2 and MCF-7 cell lines
(
Fig. 5D). Cell cycle arrest at various phases of cell division precedes apoptosis.
Treatment of HepG2 and MCF-7 cells with BER5 (GI50 concentration) for 24 h showed a
gradual increase in the number of cells in the G0/G1 phase. The percentages of G0/G1
population for the control and BER5 treated HepG2 cells were 57.3 % and 67.5 %,
respectively, and for MCF-7 were 50.5 % and 75.7 %, respectively, indicating that BER5
induced cell cycle arrest at G0/G1 phase for these two cell lines (Fig. 5E). These results
clearly suggested that BER5 induced apoptosis in HepG2 and MCF-7 cells.
Effect of BER5 induced ROS generation on apoptosis
Reactive oxygen species (ROS) generation plays an important role in apoptosis, so it is
necessary to investigate the ability of BER analogue to generate ROS. Cells were
exposed to GI50 concentration of BER5 for 24 h and analyzed for the presence of ROS by
flow cytometry. Generation of reactive oxygen species is a key factor of apoptotic cell
death. A number of studies established that berberine induced ROS generation and this
2
has been reviewed. To further elucidate whether BER5 induced intra cellular ROS
generation in HepG2 and MCF-7 cells, ROS content was measured by flow cytometry
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with H DCFDA. Treatment of BER5 at GI50 concentration with HepG2 and MCF-7 cells
2
exhibited significantly enhanced intracellular ROS levels (Fig. 6A).
Flowcytometric analysis revealed that the DCF fluorescence intensity in BER5 (GI50
)
treated cells was much higher for HepG2 and MCF-7 cells with respect to the control,
indicating a sharp increase in mean fluorescence intensity from the control to BER5
treated cells. Upon treatment with GI50 concentration of BER5, there was an increase in
intracellular ROS generation as compared with the control set after 24 h, which was
dampened upon pre-incubating with NAC. Following pre-incubation with NAC, DCF
fluorescence intensity of the control was lower with respect to BER5 treatment which
was higher for HepG2 and MCF-7 cells, (Fig. 6B). Moreover, pre-incubation with NAC
also inhibited the growth inhibitory role of BER5 (Fig. 6C). From this study it may be
inferred that BER5 induced death in HepG2 and MCF-7 cells precede via the ROS
mediated pathway.
Apoptotic cell death triggered by BER5 results in ROS dependent P53 activation
HepG2 and MCF-7 cells lines have wild type p53, which generally has a role in
apoptosis. Therefore, the role p53 activation in BER5 induced apoptosis was examined.
Treatment of HepG2 and MCF-7 cells with BER5 (GI50) resulted in increased expression
and translocation of p53 to the nucleus along with increased expression of p21 as
compared to the control (Fig. 7A). The corresponding fluorescence intensity graphs are
†
presented in Fig. S7 (ESI ). Moreover, cells that were incubated with 20 µM of pifithrin-a
(an inhibitor of p53 transactivation) for 2 h prior to treatment with BER5 showed
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inhibition in cytotoxicity by BER5, indicating that p53 may be involved in BER5
mediated apoptosis (Fig. 7C).
In order to evaluate whether ROS is involved in p53 activation or vice versa, HepG2
and MCF-7 cells were pre-incubated with NAC prior to BER5 treatment, which led to
the suppression of p53 and p21 expression (Fig. 7B) whereas, incubation with pifithrin-
a, prior to BER5 exposure failed to inhibit ROS production (Fig. 7D), indicating that
ROS is acting upstream of p53 but not vice versa. Thereby, it can be concluded that p53
induced apoptosis in these cell lines is ROS dependent.
Compound intake by BER5
From the biophysical studies presented above it was established that among the five
alkaloid analogues BER5 showed the highest affinity to DNA. Therefore, it has become
important to understand the cellular uptake and sub-cellular distribution of BER5 in
order to gain further mechanistic insights. Previous reports established that BER has
fluorescence property.30 BER5 exhibited fluorescence emission property and its
fluorescence intensity was higher than BER and other analogues. From emission
spectroscopy it was observed that upon binding to DNA the fluorescence intensity of
BER5 increased (Fig. 1B). In order to visualize BER5 directly in the HepG2 and MCF-7
cells, cells were grown onto a cover slip and treated with BER5 as described in the
experimental section, and was evaluated using a Confocal microscope at 12 h and 24 h
time periods. DAPI was used as the nuclear marker. The fluorescence intensity of the
analogue in cells was evaluated at 12 h and 24 h and it was found that at 12 h dense
fluorescence intensity around nucleus (Fig. 8A) and at 24 h fluorescence was detected
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inside the nucleus (Fig. 8B) which proved that with time BER5 enters the cell nucleus.
The uptake of BER5 was measured by treating cells for 24 h with BER5 (GI50) and
measuring using flow cytometry which indicated a higher fluorescence in both HepG2
and MCF-7 cells as compared to the control (Fig. 8C).
Conclusions
Here we have presented results on the DNA interaction of five new berberine analogues
with pyridinealkyl substitution at the 13-position of the isoquinoline chromophore. The
results obtained from absorption and fluorescence studies suggested at first that the
analogues bound to DNA stronger than berberine, and that the increased affinity is
applicable only when the linker chain length was beyond one CH group. Thus, a
2
critical length was required for effecting higher binding. The binding affinity of the
analogue BER5 was almost ten times higher than that of berberine. Unlike berberine, the
analogues bound DNA in a cooperative manner and stabilized the DNA helix strongly
against thermal strand separation. Similar results have been reported previously for
analogues with substituents at 9- and 13-positions.18,21 The binding of these analogues
have been inferred to occur through intercalation based on the results from viscosity
and fluorescence quenching experiments, and further confirmed from molecular
modeling studies. The conformational changes effected in DNA on binding and the
induced circular dichroism acquired by the analogues further substantiated their
stronger binding over berberine. From calorimetry results it was revealed that the
analogues bound to DNA by an entropy driven manner that is justified because of their
long side chain that can effect strong disruption of the water structure in the grooves of
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the DNA. Furthermore, the results of salt and temperature dependent studies proposed
that the binding mostly involved non-polyelectrolytic forces which were corroborated
from the value of the heat capacity change. Overall, the structural studies present a
remarkably stronger affinity and better interaction profile of these analogues over
berberine to DNA.
Among the five 13-pyridinealkyl berberine analogues, BER5 showed maximum
cytotoxicity towards HepG2 and MCF-7 human tumour cells after 24 h, but in different
degrees. Interestingly, 40 µM of BER5 did not show any toxic effects on normal HEK293
cells suggesting a potential therapeutic window. Significant annexin V-FITC positivity
and accumulation of cells at G0/G1 phase had indicated that BER5 induced apoptosis
in HepG2 and MCF-7 cells.
As HepG2 and MCF-7 cells are p53 wild type the role of p53 is quite relevant here. Pre-
incubation with NAC resulted in an increase in cell viability in the presence of BER5
while pre-incubation with PFT-α did not prevent ROS generation. However, p21 the
transcriptional targets of p53 was affected by the inhibitory role of NAC on ROS. BER5,
therefore, may be inducing ROS generation upstream of p53 leading to apoptosis in the
ROS mediated pathway. To conclude, BER5 mediates apoptosis in HepG2 and MCF-7
cells via a ROS mediated p53 dependent pathway and makes it a promising anticancer
agent towards p53 wild type tumors.
Materials and methods
Materials
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DNA from Calf thymus (CT, type XI, 42% guanine-cytosine composition),
.
.
poly(dA) poly(dT), poly(dG) poly(dC), berberine chloride (BER), L-glutamine, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum
(FBS), streptomycin, penicillin, Dulbecco’s modified eagle medium (DMEM), 4-2-
hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), JC-1, 6-diamidino-2-
phenylindole dihydrochloride (DAPI), PI, Annexin V-FITC, and other general reagents
were purchased from Sigma (St. Louis, USA). Antibodies against p53 and p21 were
purchased from Cell Signalling (Danvers, USA). The biophysical experiments were
performed in citrate-phosphate (CP) buffer of pH 7.4, containing 10 mM Na
2
HPO
4
.
Concentration was measured in a spectrophotometer by using molar absorption
-1
-1
2
18
coefficients (ε, in M cm ) as follows. Calf thymus DNA : 13.2x10 (at 260 nm),
2
2
poly(dA).poly(dT) : 13.6 x10 at 260 nm, poly(dG).poly(dC) : 16.8 x10 at 255 nm,
2
19
2
berberine : 22.5 x10 (at 345 nm) and all the 13-analogues: 21.3 x10 at their respective
λ
max
.
Synthesis of 13-pyridinealkyl berberine derivatives BER1-BER5
The 13-pyridinelalkyl berberine derivatives were claimed in US Pat. 8,188,109B2 to
Naxospharma srl. These were synthesized starting from commercial sample of
berberine chloride hydrate (ca. 17% H O, Shanghai Trust & We, China) and the
2
appropriate pyridine (alkyl) carboxaldehydes (Fig. 1a), via a modification of a unusual
enamine-aldehyde condensation performed on dihydroberberine31 and purified by
following the method of Weber.32 The aldehydes were prepared starting from
commercial pyridine-4-carboxaldehyde (Scheme 1b) or 4-(2-hydroxyethyl)pyridine
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(
Scheme 1c) (both purchased from Alfa Aesar GmbH & Co KG, Karlsruhe, Germany),
following usual homologation and oxidation methods and procedures known in
organic chemistry.
The purity (>94%) of the derivatives was ascertained on the Jasco HPLC LC-2000 series
system (Jasco International, Europe) with an Agilent Eclipse XDB-C18 (4.6 mm x 150
mm x 3.5 mm) column (Agilent Technologies, USA) by applying a method by Weber.
The rate of flow of the mobile phase (68% 30 mM ammonium acetate, 14 mM TEA, pH ~
4.85, 32% acetonitrile) was maintained at 1 mL/min. and absorbance was measured at
four wavelengths viz. 235, 265, 340, and 420 nm, respectively.
1
The structures of the derivatives were confirmed by H NMR spectra measured on a
Varian Mercury 400 MHz spectrometer.
1
3-[(pyridin-4-yl)methyl]-9,10-dimethoxy-5,6-dihydrobenzo[g]-1,3-benzodioxolo[5,6-
a]quinolizinium chloride, BER1: (DMSO-d6), : 10.07 (s, 1H), 8.45 (ddd, 5H), 8.10 (d,
1
H), 7.76 (d, 1H), 7.20 (d, 2H), 6.85 (s, 1H), 6.09 (s, 2H), 5.70 (s, 1H), 4.81-4.61 (t + s, 6H),
.08 (d, 6H), 3.12 (d, 2H).
4
13-[2-(pyridin-4-yl)ethyl]-9,10-dimethoxy-5,6-dihydrobenzo[g]-1,3-benzodioxolo[5,6-
a]quinolizinium chloride, BER2: (DMSO-d6), : 10.06 (s, 1H), 8.51 (d, 2H), 8.33 (d,2H),
8
3
.08 (d, 1H), 7.73 (d, 1H), 7.18 (d, 1H), 7.05 (s, 1H), 6.83 (s, 1H), 6.08 (s, 2H), 4.09 (s, 6H),
.14 (m, 2H), 2,82 (m, 2H).
13-[3-(pyridin-4-yl)propyl]-9,10-dimethoxy-5,6-dihydrobenzo[g]-1,3-benzodioxolo[5,6-
a]quinolizinium chloride, BER3: (CDCl ), : 10.35 (s, 1H), 8.52 (d, 1H), 8.05 (d, 1H), 7.88
3
2
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(
d, 1H), 7.64 (d, 1H), 6.88 (d, 2H), 6.15 (s, 1H), 5.09 (br, 2H), 4.33 (s, 3H), 4.06 (s, 3H),
3
1
.44 (br, 2H), , 3.23 (br, 2H), 2.98 (br, 2H), 2.19 (br, 2H).
3-[4-(pyridin-4-yl)butyl]-9,10-dimethoxy-5,6-dihydrobenzo[g]-1,3-benzodioxolo[5,6-
a]quinolizinium chloride, BER4: (CDCl
3
), : 10.49 (s, 1H), 8.57 (d, 2H), 7.83 (s, 2H), 7.37
d, 2H), 6.98 (s, 1H), 6.89 (s, 1H), 6.12 (s, 1H), 5.17 (br, 2H), 4.31 (s, 3H), 4.07 (s, 3H), 3.34
t, 2H), 3.18 (t, 2H), 2.80 (br, 4H).
(
(
13-[5-(pyridin-4-yl)pentyl]-9,10-dimethoxy-5,6-dihydrobenzo[g]-1,3-benzodioxolo[5,6-
a]quinolizinium chloride, BER5: (DMSO-d6), : 9.90 (s, 1H), 8.40 (d, 2H), 8.20 (d, 2H),
7
.24 (d, 2H), 7.14 (s, 2H), 6.14 (s, 2H), 4.77 (br, 2H), 4.05 (s, 6H), 3.04 (br, 2H), 2.50 (m,
H), 1.70 - 1.20 (m, 6H).
2
Methods
Spectroscopic studies
The UV-vis absorption studies used a Jasco V660 unit (Jasco International Co. Ltd.,
3
3
Hachioji, Japan). The methodology reported by Chaires and described in details earlier
was followed.30,34,35 All the fluorescence experiments were carried out in a Quanta
Master 400 unit (Horiba PTI, Canada) controlled with FelixGX spectroscopy software
provided with the instrument. The temperature was controlled by EHEIM type-2232
307 (GmbH & COKG, Germany). The alkaloid analogues were excited at their
absorption maxima near 345 nm.
CD spectropolarimetric studies
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Circular dichroism spectropolarimetric experiments used a J-815 Jasco unit (Jasco
International Co. Ltd.) equipped with a peltier cuvette holder and associated
temperature programmer as reported earlier.36-38
Optical thermal melting experiments
A Pharmaspec 1700 unit (Shimadzu Corporation) with a peltier devise (TMSPC-8) was
used for DNA melting experiments as described earlier.18 A Teflon stoppered eight
segment quartz cuvette (1 cm optical path length and 110 µl volume) was used for the
melting experiments. DNA sample (20 µM) was mixed with varying concentrations of
the alkaloid analog under study (upto D/P=1) and the mixture was heated at a rate of
0.5 °C/min, continuously monitoring the absorbance change at 260 nm. The melting
temperature (T ) is the midpoint of the transition profile as determined by the maxima
m
of the first derivative plots.
Fluorescence quenching studies
Quenching experiments were performed with the negatively charged quencher
K
4
[Fe(CN)
] as described previously18 and discussed in details in the electronic
6
supplementary information. The data were analyzed by Stern-Volmer equation and
plotted as relative fluorescence intensity versus the ferrocyanide ion concentration.
Hydrodynamic studies
The viscosity was determined by measuring the time of flow through a size 75 capillary
viscometer from Cannon Instruments Company that was submerged in a thermostated
3
7
water bath (20 ± 1 °C). Relative viscosity of DNA in the absence and in the presence of
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the alkaloid analogues was calculated by equations described in the electronic
supplementary information.
Isothermal titration calorimetry
ITC experiments were performed using MicroCal VP-ITC unit (MicroCal, LLC, USA) as
reported earlier.30,38,39 The calorimeter was calibrated from time to time as per the
manufacturer's protocols. Degassed alkaloid solutions (500 µM) were injected from the
rotating syringe into the sample chamber that housed the DNA solution. For control
experiments same protocol of injecting alkaloid into the experimental buffer was used.
The heat generated in the dilution experiment was subtracted to give the heat profile for
the DNA-alkaloid binding. The data were plotted as a function of molar ratio and fitted
to the 'one site binding model' using Origin 7.0 software to provide the thermodynamic
parameters (∆
The following equations were used to evaluate ∆
G°= -RT ln K
The binding entropy contribution (T∆
T∆ S° = ∆ H°- ∆ G°
r
H°, N and K).
r
G°.
∆
r
r
S°) was evaluated by the relationship
r
r
r
In these equations R and T represent the universal gas constant and absolute
temperature in Kelvin, respectively.
Differential scanning calorimetry
DSC experiments used a Microcal VP-differential scanning calorimeter (MicroCal, LLC).
Detailed experimental procedures have been described previously.30,40
Docking studies
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Short oligomeric DNA crystal structure of different GC- content for docking analysis,
was extracted from Protein Data Bank (PDB) identifier 258D (GC content 33.33%) and
1
JHI (GC content 66.66%). The BER5 molecule was drawn and the energy minimized by
Avogadro 1.1.1 molecular editor. Docking studies were performed with the AutoDock
.5.6 program, which utilizes the Lamarckian Genetic Algorithm (LGA). The water
1
molecules were removed from the DNA molecule used for docking. Polar hydrogen
and Gasteiger charges were added to prepare the DNA molecule for docking. Rotatable
bond in the ligand was assigned with AutoDock tools in AutoDock. The DNA and
DER5 were enclosed in the grid box that was set to 120, 120, and 120 Å along the X-, Y-,
and Z-axis with a 0.414 Å grid spacing in order to recognize the binding site of BER5 in
oligomer of 258D crystal structure and for 1JHI the grid box is 70, 60, 70 Å along the X,
Y-, and Z-axis with a 0.375 Å. Other miscellaneous parameters were assigned as per the
default values given by the AutoDock program. The lowest binding energy was chosen
from among the most possible 10 docking conformations. The output from AutoDock
was analyzed by PyMol.
Cell culture
HepG2 (human hepatocellular carcinoma), MCF-7 (human breast adenocarcinoma) and
HEK-293 (human embryonic kidney 293) cell lines were obtained from National Centre
for Cell Science, Pune, India. All cells were grown in DMEM supplemented with 10%
heat inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic, in a humidified
o
atmosphere at 37 C with 5% CO
2
.
Cell viability assay
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Cancer cells were treated in the presence of different concentrations of BER, BER1,
BER2, BER3, BER4 and BER5 (0, 10, 20, 30, and 40 µM) dissolved in CP buffer and their
respective GI50's were determined in HepG2 and MCF-7 cell lines by MTT assay as
4
0
described earlier. In another set of experiment, HEK-293 cells were treated with BER5
(
0-40 µM) for 24 h. Absorbance of the formazan was measured at 595 nm using an
ELISA reader.
Detection of apoptosis by flow cytometry
Measurement of apoptosis was performed by using an annexin V-FITC apoptosis
4
0
detection kit as described. MCF-7 and HepG2 cells were treated with BER5 (GI50) for
24 h and stained according to the protocol of the manufacturer. The % of live, apoptotic
and necrotic cells were analyzed in cell BD LSR Fortessa cell analyzer (Becton
4
Dickinson, San Jose, CA, USA). 1 x 10 cells in each sample were analyzed.
Analysis of cell cycle arrest
Cells were treated with BER5 for 24 hours. This was followed by PI staining and cell
4
0
cycle arrest analysis, as described. The percentage of cell population undergoing cell
4
cycle arrest were analyzed. 1x10 cells were analyzed for each sample.
Intracellular ROS measurements
6
Cells (1x10 ) were treated with BER5 for 24 h and the levels of intracellular H
2
O
2
were
DCFDA (20 mM) for 30 min at
7oC. For inhibition of ROS generation, cells were pretreated with Nacetyl cysteine (10
assessed by flow cytometry after incubating with H
2
3
4
41
mM) for 30 min before BER5 treatment. 1x10 cells were analyzed.
Confocal microscopy for immunocytochemistry studies
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HepG2 and MCF-7 cells were treated with BER5 (0 and GI50) for 24 h and confocal
microscopy for immunocytochemistry analysis of p53 and p21 was done as reported
4
1
earlier. In another set of experiments, cells were incubated with 2.5 mM of NAC 2 h
prior to BER5 treatment and immunocytochemistry analysis of p53 and p21 was done.
Cells were observed under an Andor spinning disk confocal microscope and images
were acquired and fluorescence intensity measured using Image J 1.46 software.
Compound uptake and intracellular localization studies of BER5 using flow
cytometry and confocal microscopy
5
To analyze quantitatively HepG2 and MCF-7 cells were seeded (1×10 for each sample)
and BER5 was added. After 24 h of incubation cells were twice washed with PBS and
analyzed using flow cytometry. To determine qualitatively, whether BER5 binds with
DNA in vitro, HepG2 and MCF-7 cells were grown on cover slips with complete
medium for 24 h and were incubated with GI50 concentration of BER5 for 12 h and 24 h
o
at 37 C. At the end of incubation period, cells were washed with cold PBS and fixed
o
with 3% paraformaldehyde for 15 min. at 4 C. DAPI (1 µg/ml) was added at room
temperature for nuclear demarcation. After washing the cells twice with PBS and they
were analyzed by confocal microscopy. Fluorescence was analyzed under an Andor
spinning disk confocal microscope. Images were acquired and fluorescence intensity
was measured using Image J 1.46 software.
Acknowledgments
This work was funded by grants from the Council of Scientific and Industrial Research
(
CSIR), Govt. of India net work project BSC0123 (GenCODE). S. Chatterjee is a Senior
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Research Fellow supported by the University Grants Commission, Government of
India. Authors express sincere thanks to Dr. S. N. Bhattacharyya, Principal Scientist and
Mr. D. Sarkar of this institute for help during confocal microscopic analysis. Ms. A.
Ganguly's assistance in flow cytometric study experiments is appreciated.
Naxospharma srl, Italy, acknowledges financial support by the Italian Ministry of
Economic Development (Grant No. 01705) under the 6th call of the EuroTransBio
initiative, transnational project BER.T.A (BERberine as antiTumour Agents).
2
9