Novel Amphiphilic G-Quadruplex Binding Synthetic Derivative of TMPyP4 and Its Effect on Cancer Cell Proliferation and Apoptosis Induction

Article abstract of DOI:10.1021/acs.biochem.8b00843

Porphyrins are well-known anticancer agents because of their high binding affinity for G-quadruplex DNA and excellent photophysical properties. Several studies carried out using TMPyP4 established it as an efficient chemotherapeutic and a photodynamic therapeutic (PDT) agent, but its use as a lead molecule has been restricted because of its high level of binding to double-stranded DNA (dsDNA), which may have side effects on normal cells and tissues. To minimize its interaction with dsDNA and to enhance internalization into cells, an analogue of TMPyP4 (5Me) was synthesized. Its selectivity for G-quadruplex DNA over dsDNA was evaluated by spectroscopic methods, and its role in stabilizing G-quadruplex DNA was assessed by fluorescence lifetime and thermal melting experiments. Biophysical studies indicated that 5Me interacts well with G-quadruplex DNA. In vitro cytotoxicity experiments with tumor cell lines (PANC-1, B16F10, and MDA MB 231) have revealed that 5Me can inhibit the growth of cancer cells comparable to TMPyP4. MTT and apoptotic assays demonstrated the ability of 5Me to specifically affect cancer cells over normal cells. Cell cycle analysis showed that 5Me, like TMPyP4, induces G2/M phase cell cycle arrest. In addition, 5Me is more effectively taken up by both cancer and normal cells than TMPyP4. In addition, we have noticed that 5Me is more efficient than TMPyP4 in inhibiting the growth of the cancer cells after irradiation with light (600-720 nm, 20 J/cm², 50 mW/cm²). By and large, these experimental results indicate that 5Me can be an efficient chemotherapeutic as well as a PDT agent.

Full text of DOI:10.1021/acs.biochem.8b00843

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Article
Novel Amphiphillic G-quadruplex Binding Synthetic Derivative of TMPyP4
and its Effect on Cancer Cell Proliferation and Apoptosis Induction
Ushasri Chilakamarthi, Devulapally Koteshwar, Sudhakar Jinka, Narra Vamsi
Krishna, Kathyayani Sridharan, Lingamallu Giribabu, and Narayana Nagesh
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00843 • Publication Date (Web): 27 Oct 2018
Downloaded from http://pubs.acs.org on October 29, 2018
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Novel Amphiphillic G-quadruplex Binding Synthetic Derivative of TMPyP4 and its
Effect on Cancer Cell Proliferation and Apoptosis Induction
Ushasri Chilakamarthi,†* Koteshwar Devulapally,† Sudhakar Jinka,‡ Vamsi Krishna Narra,† Kathyayani
Sridharan,‡ Narayana Nagesh,§* Lingamallu Giribabu †*
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†
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Polymers and functional materials Division, CSIR–Indian Institute of Chemical Technology, Hyderabad-500007, India.
‡Lipid Sciences and Technology, CSIR–Indian Institute of Chemical Technology, Hyderabad-500007, India.
§CSIR- Centre for Cellular and Molecular Biology, Hyderabad-500007, India.
Abstract
Porphyrins are well known anticancer agents due to their higher binding affinity to G-quadruplex DNA
and excellent photophysical properties. Several studies carried out using TMPyP4 established it as an
efficient chemotherapeutic as well as Photodynamic therapeutic (PDT) agent. But its use as a lead
molecule is restricted due to its high level of binding to dsDNA, which may cause side effects to normal
cells and tissues. In order to minimize its interaction with dsDNA and to enhance internalization into
cells, an analogue of TMPyP4 (5Me) is synthesized. Its selectivity to G-quadruplex DNA over dsDNA is
evaluated by spectroscopic methods and its role in stabilizing G-quadruplex DNA was assessed by
fluorescence life time and thermal melting experiments. Biophysical studies indicated that 5Me has good
interaction with G-quadruplex DNA. In vitro cytotoxicity experiments with tumor cell lines (PANC-1,
B16F10, MDA MB 231) have revealed that 5Me inhibited cancer cell growth comparable to TMPyP4.
MTT and apoptotic assays demonstrated the ability of 5Me to specifically affect cancer cells over normal
cells. Cell cycle analysis showed that 5Me is arresting the cells at G2/M phase similar to TMPyP4.
Further, 5Me is more effectively taken up by both cancer and normal cells compared to TMPyP4. In
addition, we have noticed that 5Me is more efficient than TMPyP4 in inhibiting the growth of the cancer
cells after irradiation with light (600-720 nm, 20 J/cm2, 50 mW/cm2). By and large, the present
experimental results indicate that 5Me can be an efficient chemotherapeutic as well as PDT agent.
Key words: TMPyP4; G-quadruplex DNA; spectroscopy; flow cytometry; apoptosis; PDT.
________________________________________________________________________
Corresponding author
L. Giribabu, Phone: +91-40-27191724, Fax: +91-40-27160921; E-mail: giribabu@iict.res.in
U.Chilakamarthi, Phone: +91-40-27191724, Fax: +91-40-27160921; E-mail:chusha01@gmail.com
Narayana Nagesh, Phone: +91-40-27192568; fax: +91-40-27160311; E-mail: nagesh@ccmb.res.in
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1. INTRODUCTION
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In Eukaryotes cell division is a tightly regulated process. Each stage of the cell cycle is critically
monitored by check points and the loss of this control mechanism leads to the development of cancer.
One such type of regulatory mechanisms is the replicative senescence where cell division stops due to
progressive shortening of telomeric DNA length. Telomeres are the ends of eukaryotic chromosomes and
contain long stretches of repetitive sequence TTAGGG which can form G-quadruplex (G4). The length
of telomere is maintained in the cells that need to divide regularly (germ cells and adult stem cells) by an
enzyme known as telomerase which adds telomeric repeats to the G-rich single stranded overhang.1
Cancerous cells overcome this check point by over expressing telomerase whereas majority of somatic
cells do not express telomerase.2 The inherent mechanism in DNA to inhibit telomerase activity is the
formation of G-quadruplex DNA. The telomeric G-rich region of DNA tends to form intramolecular
parallel or antiparallel quadruplex structures which prevent the binding of telomerase at this region.
Hence small molecules which can stabilize the G4 can be promising anticancer agents. This would
selectively repress the growth of cancer cells with minimal side effects to normal somatic cells.3,4
In addition, G4 motifs have been observed to locate immediate upstream of transcriptional
initiation sites of oncogenes like c-myc, Bcl-2, KRAS, c-kit etc.5 The small molecules employed to down
regulate the transcription of these oncogenes have antiproliferative activity.6-11 This is also another
intrinsic mechanism to regulate cell cycle by inhibiting the transcription of genes required for cell cycle.
Hence molecules that can bind to G-quadruplexes and stabilize it can work at two regulatory levels, one,
by stopping the cell division directly through shortening of telomeric DNA and another by acting as
transcriptional repressor of genes required for cell division. Considering the above mentioned reasons,
specifically targeting G-quadruplexes by small molecules could be a promising anticancer strategy.
Porphyrins are studied as potential photodynamic therapeutic agents for cancer treatment due to
their excellent photophysical properties.12,13 Unlike other porphyrin compounds cationic porphyrin
TMPyP4 has been reported as a chemotherapeutic agent also owing to its ability to bring about
replicative senescence by stabilizing G-quadruplex DNA at the telomeric ends.14 Like most of the
chemotherapeutic agents TMPyP4 also causes severe side effects as it has almost equal affinity towards
dsDNA and can alter the expression of genes related to cell adhesion and migration at lower
concentrations.15 Designing a molecule with high selectivity towards G-quadruplex over dsDNA that
would not interfere much with the functions of normal cells is a great challenge faced by medicinal
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chemists. Apart from this, cellular and nuclear uptake is also a major concern in the design of G4 binding
agents. It was observed that the uptake of amphiphilic molecules is more compared to hydrophilic or
hydrophobic molecules. Lipophilicity of cationic molecules can be increased either by reducing the
positive charge or by adding alkyl groups or both.16 Earlier, the effect of lipophilicity on cellular uptake
was well demonstrated where C14, an alkylated derivative of TMPyP4, is more efficiently taken up by
cells compared to TMPyP4.17,18
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Therefore, in order to improvise the potential of porphyrin molecule, it is imperative to have an
amphiphilic molecule that can differentiate G-quadruplex regions over duplex DNA. In an attempt to
modify TMPyP4 we synthesized its analogue with minimized dsDNA binding and reduced side effects to
normal cells. The rationale of our design is lowering the positive charge by one degree by replacing the
meso position N-methyl pyridyl moiety with phenyl methyl acetate. We speculate that this modification
might reduce the affinity towards dsDNA while preserving G4 binding affinity. This would also help in
increasing penetration through cell and nuclear membranes due to introduction of lipophilic methyl
acetate group, rendering the molecule amphiphilic. Further, phenyl methyl acetate is chosen not to
disturb the structure of molecule much and reduce steric hindrance during intercalation with G4 DNA.
Based on the above assumption, a synthetic analogue (5Me) was synthesized and its selectivity towards
G-quadruplex DNA and specificity towards tumor cells compared to TMPyP4 was studied.
Here we report the synthesis, purification of TMPyP4 analogue (5Me) and study of its binding
with G-quadruplex DNA using spectroscopic techniques. The affinity of 5Me to G4 and dsDNA was
validated by using synthetic oligonucleotides namely Tel Q (24 mer G-quadruplex forming oligo), Tel M
(where G11
A mutation in Tel Q disrupts G-quadruplex formation) and dsDNA.19 5Me showed G4
binding comparable to TMPyP4 (6Me) and has excellent discriminating capacity for G4 over duplex
DNA. In addition, just like TMPyP4, 5Me also has affinity towards the G4 structures formed by the
promoter regions of oncogenes such as c-myc, Bcl-2, KRAS etc. Cell culture studies for anti-
proliferative activity have shown that its tumor killing ability is comparable with TMPyP4 but at the
same time it was showing lesser effect on the growth of normal cells up to 80 µM. Because of its
specificity to G-quadruplex regions, 5Me can be a promising G-quadruplex binding anticancer agent.
Also 5Me showed better cell and nuclear internalization compared to TMPyP4. The phototoxicity
experiments indicated the efficiency of 5Me over TMPyP4 (6Me) and further investigations are in
progress. Hence 5Me can be a promising light independent as well as light dependent anticancer agent
pertaining to its dual effects.
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2. Materials and Methods
2.1 Quadruplex forming oligonucleotide, oncogene promoter regions, TMPyP4 (6Me) and 5Me.
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Synthetic oligonucleotides were purchased from Bioserve, Hyderabad. The human telomeric region
contains stretches of d(TTAGGG) sequences, that were shown to form G-quadruplex conformation. The
Tel Q or Tel M oligonucleotides and the oligonucleotides corresponding to promoter regions of
oncogene were dissolved in 10 mM Tris–HCl, 0.1 mM EDTA, and 100 mM KCl (pH 7.5). Similarly,
dsDNA was dissolved in 10 mM Tris–HCl, 0.1 mM EDTA. The DNA solutions were heated to 90 °C for
5 min and then cooled slowly to room temperature to allow them to form G-quadruplex and double
o
stranded conformation respectively. DNA samples were incubated at 4 C for 16 h and then diluted
appropriately before each spectroscopy experiment. These DNA were used for all the spectroscopic
experiments. The oligos used in this study are given in Table 1. Cell lines PANC-1, B16F10, MDA MB
231, CHO were procured from National Centre for Cell Sciences (NCCS), Pune, India. The cells were
cultured in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and
antibiotics (pencillin 100U/ml, streptomycin 100 µg/ml) solution (Sigma). The cells were grown in a
humidified atmosphere with 5% CO2 and 95% air at 37 °C. Cell culture plates and confocal dishes were
from Corning Inc., Corning, NY. All chemicals and reagents required for the synthesis of TMPyP4
(6Me) and 5Me were obtained from Aldrich (Sigma-Aldrich, St. Louis, MO, USA). MTT, PI, RNase,
DCFDA were purchased from Aldrich (Sigma-Aldrich, St. Louis, MO, USA).
Table 1. The details of oligonucleotides synthesised and used in the present study.
S.No
Name
Sequence of oligonucleotides
1.
2.
3.
Tel Q
Tel M
(TTAGGG)4
TTAGGGTTAGAG(TTAGGG)2
dsDNA
d(AC)8 · d(GT)8 or
ACA CAC ACA CAC ACA CGT GTG TGT GTG TGT GT
4.
5.
6.
Bcl2
CGG GCG CGG GAG GAAGGG GGC GGG AGC
TGG GGA GGG TGG GGA GGG TGG GGA AGG
GGG CGG TGT GGG AAG AGG GAA GAG GGG GAG
C-myc
KRAS
2.2. Synthesis and characterization of TMPyP4 (6Me) and its derivative (5Me)
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2.2.1. Preparation of 5, 10, 15, 20-Tetra(4-pyridyl)porphyrin, 1 and methyl 4-(10,15,20-tri(pyridine-4-
yl)porphyrin-5yl)benzoate, 2
The structures of the molecules and the scheme for synthesis are given in Figure 1. Porphyrin 1 and
porphyrin 2 were synthesized by Adler method with small modifications (20). 4-Pyridinecarboxaldehyde
(2.0 g, 18.67 mmol) and methyl formyl benzoate (9.2 g, 54.81 mmol) were heated in propionic acid (500
mL) at 90 °C. Freshly distilled Pyrrole (1.10 mL, 73.08 mmol) was added drop wise and the mixture was
stirred at 140 °C for 3 h. Propionic acid was removed under vacuum and the porphyrin mixture was
purified by repeated column chromatography on silica gel with Hexane/THF (10:2) as eluent. Out of the
six bands of reaction mixture, 6th and 5th bands were collected, recrystallized using MeOH/THF and
labelled as porphyrin 1 (yield 5.13 %) and porphyrin 2 (yield 7.5 %) as light purple powders.
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Porphyrin 1 1H NMR (500 MHz, CDCl3): δ = 9.08 (d, 8H, J= 4.1 Hz, o-pyridyl-H), 8.87 (s, 8H, β-pyrrole-
H), 8.18 (d, J = 4.3 Hz, 8H, m-pyridyl-H), −2.92 (s, 2H, imino-H) ppm, MALDI-TOF: m/z [M]+calcd. For
C40H26N8, 618.70; found, 619.20.
Porphyrin 2 1H NMR (400 MHz, CDCl3) : δ = 9.01 (d, J = 4.4 Hz, 6H, o-pyridyl-H), 8.89 – 8.81 (m, 8H, β-
pyrrole-H ), 8.48 – 8.40 (m, 2H, o-phenyl-H), 8.28 (s, 2H, m-phenyl-H), 8.14 (d, J = 4.5, Hz, 6H, m-pyridyl-
H), 4.11 (s, 3H, methyl), -2.88 (s, 2H, imino-H ) ppm. MALDI-TOF: m/z [M]+calcd. For C43H29N7O2, 675.95;
found, 676.20.
2.2.2. Synthesis of 4,4’,4”,4’”-(porphyrin-5,10,15,20-tetrayl)tetrakis(1-methylpyridin-1-ium) (TMPyP4
or 6Me)
Porphyrin 1 (200 mg, 0.324 mmol) was dissolved in 100 mL of N, N dimethylformamide (DMF) at 80
°C, then excess methyl iodide (about 12 mL, 194 mmol) was added and stirred for 3 h at 140 °C under
N2 atmosphere (21). The reaction was completed after refluxing for about 12 h. The DMF and excess
methyl iodide were removed under vacuum with reduced temperature to get pure TMPyP4 (6Me) (yield
67 %).
1
TMPyP4 (6Me): H NMR (500 MHz, CDCl3): δ = 8.75 (m, 8H, o-pyridyl-H), 8.45 (d, 8H, J = 4.1 Hz, β-
pyrrole-H), 8.30 (d, J = 4.3 Hz, 8H, m-pyridyl-H), 4.11 (s, 12H, N+-methyl), −2.81 (s, 2H, imino-H) ppm,
MALDI-TOF: m/z [M+H]+calcd. For C44H38N84+, 679.00; found, 680.22.
2.2.3. Synthesis of 4,4’,4”-(20-(4-(methoxycarbonyl)phenyl)porphyrin-5,10,15-triyl)tris(1-methylpyridin-
1-ium) (5Me)
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This porphyrin was synthesized by similar procedure that adopted for 6Me. (Yield 57%).
1
5Me: H NMR (500 MHz, DMSO-d6): δ = 8.91 (d, J = 4.3 Hz, 6H,o-pyridyl-H), 8.75 – 8.73 (m, 8H, β-
pyrrole-H ), 8.35 – 8.30 (m, 2H, o-phenyl-H), 8.20 (m, 2H, m-phenyl-H), 8.08 (d, J = 4.5, Hz, 6H, m-pyridyl-
H), 4.11 (s, 12H, N+-methyl, O-methyl), -2.92 (s, 2H,imino-H) ppm. MALDI-TOF: m/z [M]+calcd. For
C46H38N7 O23+, 720.95; found, 721.02.
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N
N
O
O
O
NH
N
N
Pyrrole, Propionicacid
90 0C, Reflux, 3 h
O
O
N
NH
N
N
N
N
N
HN
HN
O
N
N
Porphyrin-2
Porphyrin-1
MeI, DMF
120 0C, 12 h
MeI, DMF
120 0C, 12 h
N
N
I
I
NH
N
N
NH
N
N
O
N
N
N
O
HN
HN
I
I
I
N
N
I
I
6Me
5Me
Figure 1. The scheme for the synthesis of TMPyP4 (6Me) and 5Me.
2.3. Study of Selectivity of 5Me to G-quadruplex over dsDNA by spectroscopic methods
Spectrophotometric techniques (absorption, CD and fluorescence) are excellent tools to evaluate the
binding of small molecules to nucleic acids. Hence, we carried out absorption, circular dichroism and
fluorescence titrations to check binding affinities of 5Me and 6Me with Tel Q, Tel M and dsDNA.
2.3.1. UV-Visible titrations to find out the affinity of 5Me with different types of DNAs Binding assays of
5Me and 6Me were performed with the above prepared Tel Q, Tel M and dsDNA (23). Aliquots of Tel Q
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(0-25 µM) or Tel M (0-20 µM) were added step wise to a constant concentration of 5Me (5 µM).
Similarly, aliquots of Tel Q (0-40 µM) or Tel M (0-32.5 µM) were added step wise to 6Me (5 µM) until
saturation. In a similar mode, double stranded DNA (0-30 µM) and (0-50 µM) were added to 5Me (10
µM) and 6Me (10 µM) respectively. After each addition, the mixture was incubated for 5 min and
absorption spectra were recorded with Shimadzu UV-3600, UV-visible-NIR spectrophotometer.
Recording was stopped when the three successive additions resulted in no change in absorbance profile.
Percentage hypochromicity and bathochromic shift were calculated from the titration data. The percent
hypochromicity of soret band was calculated using the equation given below.
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The percent hypochromicity = [(ɛf - ɛb)/ɛf] x100, where ɛb=Ab/Cb
Binding constants of 6Me and 5Me with all the three types of DNA were calculated following an earlier
study.22,23 In a similar manner, UV titrations of 5Me were carried out with the promoter sequences of
oligonucleotides of oncogenes c-myc, Bcl-2 and KRAS.
2.3.2. Circular Dichroism titrations to observe changes in DNA conformation after binding with 5Me
Circular dichroism spectroscopy profiles were recorded using a JASCO 815 CD Spectropolarimeter
(Tokyo, Japan) to observe the changes in DNA conformation (24) after the interaction of TMPyP4 (6Me)
or 5Me with both G4 DNA as well as dsDNA. To a constant concentration of Tel Q or Tel M or dsDNA
(10 µM) in a 1 mm path length cuvette, increasing amounts of TMPyP4 (6Me) and 5Me (final
concentrations of 15 µM, 30 µM, 60 µM) were added sequentially and the spectra were recorded from
200 to 450 nm. The change in the profiles would indicate the interaction of TMPyP4 (6Me) or 5Me with
the respective DNA.
2.3.3. Fluorescence titrations to find out the affinity of 5Me to different types of DNAs
Steady-state fluorescence spectra of 5Me were recorded using a Fluorolog-3 spectrofluorometer (Spex
model, JobinYvon) with 1 cm path length quartz cell. Tel Q or Tel M or dsDNA were added step wise to
a constant amount of 5Me (2 µM) until no increase is observed in the spectra. After 5 min incubation,
the fluorescence profiles were recorded by exciting the complex at 422 nm and emission spectra was
recorded from 600-800 nm. The DNA was added until there was no change in the fluorescence profile
observed.
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2.4. Investigating the stabilizing capability of 5Me on G-quadruplex over dsDNA
Uv-vis and fluorescence thermal melting profiles indicate the stabilization or destabilization of DNA by
small molecules. In addition to this fluorescence life time also provides valuable information regarding
stabilization of DNA by small molecules. Hence thermal melting and fluorescence life time studies were
carried out.
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2.4.1. Finding out melting temperature of DNA and bound complexes by Uv-vis thermal melting
Melting studies of Tel Q, Tel M and dsDNA (25µM) were carried out in the presence and absence of
5Me or 6Me (5µM). Melting studies were performed using ABI Lambda Spectrophotometer (Waltham,
MA, USA) attached to a thermal controller. The DNA was taken in 0.1 cm path length quartz cuvette and
absorbance (A260) was recorded with increasing temperatures initially in the absence and later in the
presence of 5Me or 6Me. The DNA or complexes were subjected to increasing temperatures from 20 °C
to 80 °C with increments of 1 °C / min. From the data, melting temperatures of DNA and complexes
were derived.
2.4.2. Finding out melting temperature of bound complexes by fluorescence thermal melting
The fully saturated complexes of 5Me with Tel Q, Tel M and dsDNA (5Me: DNA =1:3) were subjected
to increasing temperatures from 20 °C to 80 °C with increments of 1 °C / min. Data was recorded after
every 2 °C. From the data Tm was calculated to find out the stabilizing effect of 5Me on the three types
of DNAs.
2.4.3. Fluorescence life time to find out the stability of 5Me complexes with different DNAs
The fluorescence decay profiles of the complexes of 5Me with Tel Q or Tel M or dsDNA were recorded
on a picosecond time-correlated single photon counting (TCSPC) setup (FluoroLog3-Triple Illuminator,
IBH Horiba JobinYvon) employing a picosecond light emitting diode laser (NanoLED, λex ~ 450 nm) as
an excitation source. This method is very sensitive for the excited state interactions between DNA and
ligand25 and hence this experiment is performed to investigate which DNA forms stable complex with
5Me (5Me: DNA =1:3). The decay curves were recorded by monitoring the fluorescence emission
maxima of the complex (λem ~ 650 nm). Photomultiplier tube (R928P, Hamamatsu) was employed as the
detector. The lamp profile was recorded by placing a dilute solution of Ludox in water in place of the
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sample. Decay curves were analyzed by nonlinear least-squares iteration procedure using IBH DAS6
(version 2.3) decay analysis software. The quality of the fits was judged by the χ2 values and distribution
of the residuals.
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2.5. Cell culture studies to investigate the efficiency of 5Me on cancer cell lines
2.5.1. Cellular uptake and Localization of the compounds TMPyP4 (6Me) and 5Me
As these (5Me and 6Me) molecules emit red fluorescence when excited at 488 nm, FACS and Confocal
microscopy observations were carried out to check cellular uptake and nuclear localization of the
compounds. Cellular uptake of TMPyP4 (6Me) and 5Me were studied by FACS in PANC-1, B16F10 and
CHO cell lines. 20 µM of TMPyP4 (6Me) or 5Me were added to culture plates in dish and kept in
incubator for 3 h. Then the cells were detached from plates using trypsin and washed with PBS twice.
Then cells were re-suspended in PBS and analyzed on FACS machine.16,17 20 µM concentrations of 5Me
and 6Me were added to cultures in confocal dishes and after 3 h of incubation Hoechst stain was added
and kept for 1 h in incubator at 37 °C. After incubation cells were washed twice with PBS. Then PBS
was added to dishes and observed under confocal microscope to check the localization of the compounds.
2.5.2. Anti proliferative activity of TMPyP4 (6Me) and 5Me in normal and cancer cell lines
MTT assay was performed to verify the cytotoxicity of 6Me and 5Me on cancer and normal cell lines.
5000 cells were seeded in each well of 96-well plate and incubated at 37 °C for 24 h. Different
concentrations of 6Me or 5Me were added in triplicates and incubated for 48 h at 37 °C. Then 10 µl of 5
mg/ml concentration of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Dipheyltetrazolium bromide) was added
to each well and further incubated for 4 h more till purple formazan crystals were formed.26 The crystals
were dissolved in DMSO and absorbance of each well was recorded at 570 nm using BioTek Synergy H1
Multi-mode Microplate Reader. The data was analyzed using GraphPad Prism 6 software.
2.5.3. Apoptotic assay to investigate the efficiency of 5Me in causing death of cancer cells
PANC-1 and CHO (1.5x105) cells were seeded in each well of a six well plate and after 24 h of
incubation at 37 °C they were treated with different concentrations of 6Me and 5Me and incubated at 37
°C for another 24 h.27 Then the cells were trypsinized, washed twice with PBS and apoptosis assay was
performed by using Biolegends apoptosis detection kit following the manufacturer’s instructions.
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2.5.4. Cell cycle Analysis
PANC-1 (1.5x105) cells were seeded in each well of a six well plate and incubated at 37 °C for 24 h. The
cells were then treated with 80 µM 5Me and harvested after 24 h, washed with phosphate buffered saline
(PBS) twice, fixed with 70% ethanol for 2 h at 4 °C. Then the cells were washed with PBS and re-
suspended in 1 ml staining buffer containing 100 µg/ml RNase A, 50 µg/ml PI, 0.1% Triton X-100. The
samples were incubated in dark for 1 h at room temperature. Then cell cycle profile was obtained with
FACS Calibur, BD Biosciences Immunocytometry Systems, San Jose, CA.28
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2.5.5. ROS generation
ROS detection using DCFDA by flow cytometry is carried out29 under dark conditions to check any ROS
generation occurring after treatment with 5Me and 6Me. 1.5x105 cells were seeded in each well of 6 well
plates and incubated for 24 h. Then different concentrations of 6Me or 5Me were added and incubated at
37 °C for 24 h. Subsequently, DCFDA was added to each well and incubated for 3 h, after that the cells
were trypsinized and washed with PBS twice. The cells were then resuspended in PBS and analyzed
using FACS machine.
2.5.6. Phototoxicity of 5Me and TMPyP4 (6Me)
Light dependent cytotoxicity of 5Me was assessed by MTT assay. Different concentrations (0-20 µM) of
5Me or TMPyP4 (6Me) were added to SKOV3 and PANC-1 cells in triplicates. After 3 h of incubation at
37 °C the cells were irradiated with Waldman 1200 PDT L system fitted with 1200 W lamp (600-720 nm
and 20 J cm-2; 50m Wcm-2), media changed and incubated at 37 °C for 24 h. Then 10 µl of MTT (5
mg/ml) was added and incubated again for 4 h at 37 °C for the formation of formazan crystals. Then the
crystals were dissolved in DMSO or isopropyl alcohol and the absorbance readings were taken at 570
nm12 using Biotek Synergy H1 Multi-mode Microplate Reader. The data was analyzed using Graphpad
Prism6 software.
3. RESULTS
3.1. Study of Selectivity of 5Me to G-quadruplex over dsDNA by spectroscopic methods
3.1.1. UV-Visible titrations to find out the affinity of 5Me with different types of DNAs
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UV-visible spectroscopy is a commonly employed technique to observe the interaction of small
molecules with DNA. In general, intercalation of the molecule in DNA results in hypochromicity and
bathchromic shift of the absorption spectra. TMPyP4 (6Me) gives strong soret band at 422 nm and four
weak Q bands at 520, 558, 584, 638 nm respectively, whereas 5Me gives strong soret band at 422 nm
and four weak Q bands at 520, 558, 598 nm and 647 nm respectively. Both compounds showed
hypochromicity and bathochromic shift at the soret band upon addition of either of the DNA species
(Figure 2A). The hypochromicity values of 5Me with Tel Q, Tel M and dsDNA are 55%, 51% and 50%
respectively, whereas TMPyP4 (6Me) exhibited the values of 74%, 70% and 59% with Tel Q, Tel M and
dsDNA respectively. The bathochromic shift for 5Me with Tel Q, Tel M and dsDNA are 15 nm, 13 nm
and 7 nm respectively, whereas the values for 6Me are 15.4 nm, 14.3 nm and 17.2 nm. The extent of
bathochromic shift is proportional to the strength of interaction. The red shift obtained by TMPyp4
(6Me) after interaction with dsDNA is more compared to that of Tel Q (Table 2). 5Me has shown very
less bathochromic shift with duplex DNA retaining almost the same bathochromic shift with Tel Q. This
signifies that the structural modification to yield 5Me has led to the decrease in interaction with dsDNA.
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Wavelength nm.
Wavelength nm.
Figure 2. (A) Comparing the affinities of 5Me and 6Me with Tel Q, Tel M and dsDNA using UV-visible
spectroscopy. The absorption profiles (i) and (ii) were obtained by the sequential addition of Tel Q (0-25
µM) or Tel M (0-20 µM) to a fixed concentration of (5 µM) of 5Me. (iii) The spectra obtained when
dsDNA (0-30 µM) was added to 10 µM of 5Me. The absorption profiles (iv) and (v) were obtained by
the sequential addition of Tel Q (0-40 µM) or Tel M (0-32.5 µM) to a fixed concentration of (5 µM) of
TMPyP4 (6Me). (vi)The spectra obtained when dsDNA (0-50 µM) was added to 10 µM of TMPyP4
(6Me). (B) UV titration graphs of 5Me with (i) c-myc (ii) Bcl-2 (iii) KRAS. The arrows show the
direction of movement of peaks upon addition of DNA.
From the bathochromic shift values it is clear that 6Me is having more affinity to dsDNA30 whereas 5Me
showed reduced affinity to dsDNA. The binding constants were calculated from the plots drawn r vs r/cf.
The constant (K) for 5Me with Tel Q, Tel M and dsDNA are 6x107 M-1, 1x107 M-1and 1x107 M-1
respectively. The binding constants obtained for TMPyP4 (6Me) are 8x107 M-1, 3x107 M-1 and 4x107 M-
1
with Tel Q, Tel M and dsDNA respectively. Overall the UV titration results indicate that 5Me is
capable of discriminating G4 over dsDNA. However, lowering the positive charge has shown little effect
on the interaction with G4 but it has hampered the binding ability with dsDNA significantly. In a similar
manner, the titrations of 5Me with the DNA sequences of promoter regions of oncogenes c-myc, Bcl-2
and KRAS were performed and the profiles were presented in Figure 2B. These profiles also showed
hypochromicity and bathochromic shift indicating its affinity towards the promoter regions of those
oncogenes. The promoter regions of oncogenes tend to form G-quadruplexes to prevent the expression of
these genes in the absence of cell division. Thus the molecule that interacts or binds these promoter
sequences can down regulate the expression of genes required for cell division and thus can have
anticancer activity.
Table 2. Details of hypochromic and bathochromic shifts obtained in UV-visible spectroscopy profiles
of 5Me and 6Me after binding with Tel Q, Tel M and dsDNA
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Compound
5Me
DNA
Initial
λmax, nm
Final
λmax, nm
Bathochro
mic Shift, nm
Hypochromicity
%
Binding
Constant
M-1
Tel Q
422
437
15
55
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50
74
70
59
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1x107
1x107
8x107
3x107
4x107
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Tel M
dsDNA
Tel Q
422
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14
422
429
7
TMPyP4
422.5
422.2
422.5
437.9
436.5
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15.4
14.3
17.2
Tel M
dsDNA
3.1.2.Circular Dichroism titrations to observe changes in DNA conformation after binding with 5Me
UV-visible titrations indicated the affinity of 5Me to G-quadruplex over dsDNA whereas 6Me has
almost equal affinity to both. To further confirm the effect of binding on the conformation changes of Tel
Q or Tel M or dsDNA, CD spectroscopy studies were carried out. Tel Q oligos showed characteristic G-
quadruplex conformation with a positive peak around 290 nm, shoulder peak at 265 nm and a negative
peak around 240 nm.31 Upon addition of TMPyP4 (6Me) or 5Me the position of signature peaks was
conserved but there was decrease in the intensity of positive peak suggesting interaction of molecule with
the G-quadruplex DNA (Figure 3 A and D). The extent of decrease in intensity is little less for 5Me
compared to 6Me, indicating reduced affinity which is also reflected from the binding constants obtained
from UV titrations. There is no significant change in the conformation of Tel M upon binding with 5Me
but with increasing concentrations of TMPyP4 (6Me)
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Figure 3. Circular dichroism profiles of 10 µM of each DNA (Tel Q, Tel M, dsDNA) in the absence or
presence of 5Me or TMPyP4 (15 µM, 30 µM, 60 µM). The conformational changes of Tel Q, Tel M and
dsDNA after interactions with 5Me is depicted in A, B and C respectively. Similarly, the conformational
changes of Tel Q, Tel M and dsDNA after interactions with TMPyP4 are depicted in D, E and F
respectively.
the positive peak at 260 nm increased (Figure 3 B and E). TMPyP4 (6Me) has shown more affinity
towards dsDNA whereas the structural modification in 5Me resulted in significant reduction in the
affinity to dsDNA (Figure 3 C and F). Induced CD (ICD) spectra were observed in the soret region with
a negative peak and the intensity of the peak increased with increased concentration of DNA.32, 33 The
increase in peak intensity is similar for both TMPyP4 (6Me) and 5Me in the case of Tel Q but with the
two other DNAs the increase is more pronounced for TMPyP4 (6Me) compared to 5Me. This indicates
that 5Me is showing less affinity to Tel M and dsDNA.
3.1.3. Fluorescence titrations to find out the affinity of 5Me to different types of DNAs
The emission spectra of 5Me were compared in the presence and absence of different DNA species.
When excited at 422 nm 5Me showed very less fluorescence. This may be because of solvent (water)
effect which might be quenching the fluorescence exerted by 5Me molecules. Upon addition of either Tel
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Q, Tel M or dsDNA the fluorescence signal kept on enhancing gradually34, 35 and finally two peaks
appeared at 660 and 720 nm (Fig 4). The extent of increase was much lower in case of duplex DNA
(Figure 4C) compared to Tel M (Figure 4B) and Tel Q (Figure 4A). G-quadruplex DNA exerted strong
effect in increasing the fluorescence of 5Me compared to other DNA species. Fluorescence data also
indicated its selectivity towards G4 over dsDNA. Then, to investigate the stabilizing ability of 5Me, we
performed fluorescence thermal melting and fluorescence life time experiments.
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Figure 4. Fluorescence titration profiles of 5Me with A) Tel Q B) Tel M and C) dsDNA. Upon addition
of each DNA species fluorescence of 5Me is increasing but the extent of attenuation is more in case of
Tel Q (D) indicates the G4 DNA selectivity of 5Me.
3.2. Investigating the stabilizing capability of 5Me on G-quadruplex over dsDNA
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The DNA stabilizing capacity of molecules can be studied from thermal melting and fluorescent life time
experiments.
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3.2.1. Finding out melting temperature of DNA and bound complexes by UV-vis thermal melting
UV-vis and fluorescent thermal melting of Tel Q, Tel M and dsDNA were carried out in the absence and
presence of 5Me and 6Me to find out the melting temperatures of DNA and their complexes with these
molecules. The melting temperatures derived from the absorption profiles were given in the Table 3 and
the absorption profiles are given in SF. 3. The increase of Tm after complexing with the molecule
indicates its stabilizing capacity. In other words more the melting temperature of the complex more is
ability of molecule to stabilize DNA. From the below table it is evident that 5Me stabilizes G-
quadruplex DNA compared to dsDNA.
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Table 3. DNA Melting temperatures in the absence and presence of 5Me or 6Me. Increase in Tm
indicates the stabilizing capability of the molecules
DNA
Tm (° C)
Increase in Tm
Δ Tm (° C)
Tel Q
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Tel Q + 6Me
Tel M
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dsDNA
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12
3.2.2. Finding out melting temperature of bound complexes by fluorescence thermal melting Thermal
melting experiments were carried out to find out the stabilizing effect of 5Me on Tel Q, Tel M and
dsDNA. Fluorescence profiles were recorded at every 2 °C increase in temperature. Graphs were plotted
with dF/dt vs temperature to obtain Tm (Fig. 5A). The Tm values of Tel Q, Tel M and dsDNA bound to
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5Me are 40 °C, 37 °C and 31 °C respectively. Thus, thermal melting experiments support the results of
earlier experiments that 5Me interacts and stabilizes Tel Q compared to dsDNA.
3.2.3. Fluorescence life time to find out the stability of 5Me complexes with different DNAs
The excited state life time decay profiles of 5Me complexes with Tel Q, Tel M and dsDNA are shown in
Fig. 5B. The complexes exhibited bi-exponential decay and the corresponding data is summarized in
table 4. The complex of 5Me with Tel Q has shown high life time, whereas with dsDNA showed less and
the complex with Tel M exhibited intermediate value. The life time values are represented in Table 4.
These results further reveal that 5Me stabilizes Tel Q compared to Tel M and dsDNA.36 Thus, the
spectroscopic data results are giving clear indication that 5Me is showing lesser affinity to dsDNA
compared to TMPyP4 (6Me). Hence, we proceeded to cell culture studies using both cancerous and
normal cell lines to check if 5Me can differentially act on normal and cancer cells.
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Figure 5. A) Thermal stability of the complexes of 5Me with Tel Q, Tel M and dsDNA (5Me: DNA
=1:3) depicted in a plot as a function of temperature. B) Fluorescence decay life times of the complexes
of 5Me with Tel Q, Tel M and dsDNA (5Me: DNA =1:3) in water.
Table 4. Fluorescence lifetime data after binding with Tel Q, Tel M and dsDNA
Complex
5Me
Tel Q
Tel M
dsDNA
1
2
1
2
1
2
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9
2.26
(52.96)
9
2.26
(52.96)
9
(52.96)
(47.04)
(47.04)
(47.04)
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5Me:DNA
(3:1)
4.21
(7.05)
16.8
(92.95)
3.97
(9.45)
15.9
(19.05)
3.72
(14.26)
14.9
(85.74 )
6
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3.3. Cell culture studies to check uptake, localization and killing efficiency of 5Me in cancer and normal
cell lines
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3.3.1. Cellular uptake and Localization of the compounds 6Me and 5Me
The compound 6Me is hydrophilic in nature while 5Me becomes amphiphilic by lowering one positive
charge and introducing lipophilic methyl acetate at one of the four meso positions of the porphyrin
molecule. The structural modification in 5Me is expected to result in its high cellular uptake in cancer
(PANC-1) as well as normal cell lines (CHO) compared to 6Me which was confirmed by the uptake
studies carried out by FACS after 3 h treatment
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Figure 6. A) Cellular uptake of 5Me and TMPyP4 (6Me) in i) PANC-1 and ii) CHO cells studied by
flow cytometry. Confocal microscopic images showing the cytoplasmic and nuclear localization of 5Me
and TMPyP4 (6Me) in B16F10 (B) and PANC-1 (C) cell lines.
of the cells with 20 µM of each compound (Figure 6A i and ii). The shift of the peak towards right
indicates uptake and the shift is more in case of 5Me compared to TMPyP4 (6Me) in both cancer (Figure
6A i) and normal cell lines (Figure 6A ii). Further we extended our studies to show qualitative
distribution of 5Me and 6Me by confocal microscopy. The high cellular and nuclear uptake of 5Me can
also be appreciated by confocal microscopic images taken after 3 h of treating the B16F10 and PANC-1
cells with 20 µM of each compound (Figure 6B and 6C). A particulate distribution with in cytoplasm is
observed for TMPyP4 (6Me) after 3 h but 5Me has almost reached nucleus by that time which is clearly
visible in merged images.
3.3.2. Anti proliferative activity of TMPyP4 (6Me) and 5Me in cancer cell lines
MTT assay was carried out to investigate the cell viability on different cancer (MDA MB231, PANC-1,
B16F10) and normal (CHO) cell lines by using 5Me and 6Me (Figure 7A and 7B). The IC50 values of
5Me lies in the range of 80-100 µM for cancer cells but has less effect on normal cells. Though the IC50
values of 6Me are less than that of 5Me, it is causing more pronounced cell death not just in cancer cells
but also in normal cells (7C). Our results are in line with the previous report saying that in vitro
cytotoxicity of 6Me is comparable in cancer and normal cells.37 Apart from G-quadruplex binding, 6Me
has more affinity to dsDNA also which may be the main contributing factor for the death of normal cells.
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5Me being more selective to G-quadruplex is more specific to cancer cells. Structural modification in
5Me has resulted in decreased binding affinity to both G-quadruplex and dsDNA, which resulted in
increase of IC50 values against cancer cells but decreased toxicity towards normal cells. A little
compromise in IC50 concentration in the case of 5Me is rewarded with the differential toxicity between
tumor and normal cells. Having established its discriminating capacity of cancer cells over normal cells,
the role of these molecules in apoptosis induction is studied using apoptotic assay and cell cycle analysis.
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Concentration of porphyrin (M)
Figure 7. Cytotoxicity of 5Me (A) and TMPyP4 (B) in PANC-1, B16F10 and MDA MB 231 cell lines.
C) Cytotoxicity of 5Me and TMPyP4 (6Me) in CHO cell line.
3.3.3. Apoptotic assay to investigate the efficiency of 5Me in causing death of cancer cells
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Figure 8. Dot plots representing the percentage of apoptotic cells in A) PANC-1 and B) CHO cell lines
analyzed by flow cytometry after the treatment with different concentrations of 5Me and TMPyP4 (6Me)
for 24 h Q1- Necrotic cells, Q2-Late apoptotic cells, Q3- Live cells, Q4- early apoptotic cells. A i) and B
i) are untreated controls, A ii) and B ii) are treated with 40 µM, A iii) and B iii) are treated with 80 µM,
A vi) and B vi) are treated with 100 µM of 5Me for 24 h A v) and B v) are treated with 40 µM, A vi) and
B vi) are treated with 80 µM, A vii) treated with 100 µM and B vii) treated with 120 µM of TMPyP4
(6Me) for 24 h.
Apoptotic assay was carried out using Annexin V - FITC detection kit on cancer (PANC-1) and normal
(CHO) cell lines after 24 hr treatment of 6Me or 5Me. Percentage of apoptotic cells after 5Me treatment
at 40 µM, 80 µM and 100 µM on PANC-1 are 11.6%, 15.2% and 27.3% respectively and that of 6Me are
25.1%, 34.1% and 52.7% respectively, whereas 8% apoptosis was observed in untreated controls of
PANC-1 (Fig. 8A) and CHO cells (Fig. 8B). The structural modification resulted in less number of
apoptotic cells for 5Me compared to TMPyP4 (6Me) which may be due to reduced binding to G-
quadruplex DNA. Interestingly, 5Me induced significantly less apoptosis in CHO cells compared to
TMPyP4 (6Me) which may be due to reduced affinity to dsDNA. The viable CHO cells (quadrant Q3 in
Figure 8 B ii,iii and iv) remained almost constant even with the increase in concentrations of 5Me. It was
observed that 6Me induced necrosis in CHO cells also. These results indicate that 5Me had low
cytotoxicity on normal cells but have significant toxicity towards cancer cells.
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G1%
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Figure 9. Cell cycle analyses showing the effect of 5Me on PANC-1 cells after 24 h. A) Untreated
control B) cells treated with 80 µM 5Me C) Graph showing percentage of cells in each phase with and
without treatment of 5Me. The cell cycle analysis was performed by propidium iodide staining method.
Cell cycle analysis was performed to find out the effect of 5Me on different phases of cell cycle. G4
binding molecules especially 6Me has shown to arrest cell cycle G2/M phase.29 In a similar way 5Me has
also shown cell cycle arrest at G2/M phase (Figure 9). The untreated control PANC-1 cells showed the
following distribution of cells in different phases (G1-47.08%, S-21.74%, G2/M-31.18%). After
treatment with 80 µM 5Me for 24 h, it was observed that 5Me was causing decrease in the percentage of
G1 cells and increase in percentage of cells in S and G2/M phases (G1-43.3%, S-22.48%, G2/M-
34.82%). It indicates that 5Me was causing growth arrest at G2/M phase similar to 6Me.
3.3.5. ROS generation
In order to understand the extent of ROS generation by 6Me and 5Me in various cancer and normal cells,
ROS detection is done by DCFDA method as reported earlier.29 Both TMPyP4 (6Me) and 5Me did not
produce any ROS at 16 h in PANC-1 cells but 6Me treatment generated slightly or insignificant amount
of ROS after 24 h in PANC-1 and in CHO after 16 h. This indicates that 5Me is not harmful to normal
cells (SF. 2).
3.3.6. Phototoxicity of 5Me and TMPyP4 (6Me)
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As 5Me is not detrimental to normal cells compared to TMPyP4 (6Me), we further checked its
phototoxicity on SKOV3 and PANC-1 cells. Interestingly, the MTT assay performed after irradiation
with light indicated that 5Me can be a potential PDT agent. The IC50 values obtained are 2.3 µM and 3
µM for SKOV3 and PANC-1 cells respectively whereas the values for TMPyP4 (6Me) are 20 µM
(Figure 10). These results indicate that though 5Me could inhibit growth of cancer cells through light
independent mechanism at higher concentrations and can bring about the same effect at lower
concentrations through light dependent mechanism.
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Figure 10. Phototoxicity of 5Me and TMPyP4 (6Me) on (A) SKOV3 and (B) PANC-1 cell lines. After
3hr of treatment with 5Me, the cells were irradiated with (600−720 nm, 20 J cm−2, and 50 mW cm−2)
light and MTT assay was performed after 24 h of incubation in new media.
4. DISCUSSION
Small molecules that show high affinity to G-quadruplex DNA act as potential anticancer molecules.
TMPyP4 (6Me) is an efficient G4 binding molecule but it failed to discriminate normal and cancer cells.
A recent study showed that TMPyP4 (6Me) at low concentrations caused cell migration and metastasis
and alters the expression of many genes which disrupts the functions of normal cells.14 Hence, we
conducted this study to have a molecule which can discriminate cancer cells from normal cells. Cellular
uptake of small molecules depends on their polarity and size. Simple diffusion across membranes is
governed by the balance between hydrophilic and hydrophobic nature of the molecules. Hydrophobic
molecules remain bounded to the cell membrane whereas hydrophilic molecules cannot cross the lipid
membrane. Therefore, amphiphilic molecules are gaining importance in drug discovery. Electrostatic
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interactions play a major role in the binding of cationic molecules to any type of DNA. Positive charge is
a necessary feature of DNA binding molecules to interact with the negative phosphate groups of DNA
back bone. In another study, lowering the positive charge on 6Me molecule resulted in decreased binding
affinity to G4 but increased selectivity.38 In this study, they replaced N-methyl pyridyl group with phenyl
moiety (named 4P3) and it has shown superior selectivity to G4 compared to TMPyP4. In our study, we
replaced N-methyl pyridyl group with phenyl methyl acetate, a lipophilic group making our molecule
5Me amphiphilic for better cell and nuclear permeability.
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The UV titration of Tel Q, mutant Tel M and dsDNA showed that 5Me retained significant
binding with G4 DNA and drastically reduced binding with dsDNA (Figure 2). The titration profiles
showed hypochromicity and bathochromic shift upon binding with all the three types of DNA but the
values observed in the case of 5Me with G4 DNA and dsDNA are indicating its selectivity to G4 over
duplex DNA (Table 2). There is increase in the fluorescence of 5Me by the addition of increasing
concentrations of DNA (Tel Q, Tel M, dsDNA). The increase was more pronounced in case of Tel Q
DNA compared to Tel M and dsDNA (Figure 4).
To further confirm the binding affinities of 5Me with the above mentioned DNA samples, the
absorption and fluorescence of the bound complexes was observed as a function of temperature. As
expected in both the experiments, the complex of 5Me with G4 DNA was more stable upon increasing
the temperature from 30 °C - 80 °C. Similarly, the fluorescence life times of the complexes also
confirmed that 5Me stabilizes G4 DNA over dsDNA (Figure 5B). The CD experiments also confirmed
its binding to G-quadruplex. Both 6Me and 5Me showed conservation of the peaks with G4 DNA or
Mutant G4 DNA or dsDNA suggesting the external binding mode. But both the molecules show induced
CD spectra at soret region by forming a negative peak with increasing ratio (r = 1.5, 3 and 6) of
porphyrin / DNA indicating intercalation (Figure 3). The hypochromicity and large bathochromic shift
in UV–vis titrations, hypochromicity and negative induced CD band at soret region in CD titrations and
hyperchromicity in fluorescence titration indicates intercalation as the preferred mode of binding.23
Based on the above results we speculate that 5Me may interact with DNA through intercalation.
From the figure, it is clearly visible that in case of 5Me the interaction with G-quadruplex DNA is
reduced to some extent, whereas with the dsDNA there is significant decrease in the binding affinity. The
parent molecule TMPyP4 is a planar molecule and the planarity of the 5 Me molecule is disturbed due to
the replacement of the meso position N-methyl pyridyl moiety with phenyl methyl acetate. Since the
plane of the molecule is disturbed, 5Me has reduced interaction with quadruplex DNA compared to 6
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Me. Further, the change made in the modified derivative (5Me) has brought down the overall positive
charge on the modified derivative (5Me). It is evident that DNA is negatively charged due to the
presence of phosphates in its back bone. Among dsDNA and quadruplex DNA, dsDNA has relatively
more negative charge compared to quadruplex DNA. Hence lowering the positive charge reduced the
interaction with dsDNA. It is reported earlier that the differential interaction of a tricationic molecule is
due to the differences in electrochemical behavior of G-quadruplex and dsDNA.40 In this paper they
discussed that non electrostatic interactions play major role in the strong association of tricaionic
hexaammineruthenium(III) cation with G-quadruplex whereas the association with dsDNA is purely
electrostatic interaction. Hence probably in the case of 5Me also lowering the positive charge by one
degree compared to TMPyP4 has shown more impact on the interaction with dsDNA compared to G-
quadruplex DNA. The same is reflected in the cytotoxicity experiments where 5Me has shown
significant toxicity on tumor cells and lesser toxicity on normal cells compared to 6Me (Figure 7).
The uptake experiments investigated by FACS showed that 5Me is more permeable into cells compared
to TMPyP4. Earlier studies with C14, a lipophilic derivative of TMPyP4 also supports this observation of
increased cellular uptake due to amphiphilic nature of the molecule.16,17 Confocal microscopy
observations indicated that higher amount of 5Me reached nucleus after 3 h treatment whereas TMPyP4
is still mostly in the cytoplasm (Figure 6B and C).
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5Me showed cytotoxicity on tumor cells and IC50 values are comparable to TMPyP4. A little
compromise with IC50 values due to structural modification of lowering positive charge resulted in low
toxicity to normal cells. Even though, the apoptotic assays indicated that 5Me is less efficient in killing
cancer cells compared to 6Me, its cytotoxic effect on normal cells is significantly low when compared to
6Me (Figure 8). TMPyP4 was shown to arrest cell division at G2/M phase29 and in similar manner 5Me
was also showing growth arrest at G2/M phase (Figure 9). We investigated ROS generation using
DCFDA method as TMPyP4 generates ROS after exposure to light, but in the dark it does not generate
ROS. The detection of ROS generation by TMPyP4 (6Me) and 5Me was carried out under dark
conditions by DCFDA method revealed that there was no ROS after 16 h and slight or insignificant
amount of ROS generated after 24 h. The cytotoxicity experiments performed under dark conditions
revealed that the IC50 values of 5Me fall in the range from 80-100 µM on different cancer cell lines.
This assay results with DCFDA, indirectly indicate that the cell death in light independent mechanism is
probably due to G quadruplex binding but not because of ROS. Further, the results of MTT assay
performed 24 h after exposure to light indicated IC50 values of 2.3 and 3 µM for 5Me treated SKOV3
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and PANC-1 cells respectively, but there was no toxicity in dark with these concentrations. Hence we
propose 5Me as a promising PDT agent and further studies are in progress.
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Earlier studies had shown that TMPyP4 could prevent the expression of oncogenes by binding to
the G-quadruplex promoter regions of those genes there by arresting the cell division. 5Me is also
showing excellent binding capacity to the promoters of c-myc, KRAS and Bcl-2 that form G-quadruplex
conformation (Figure 2B). The UV titrations showed hypochromicity and bathochromic shift upon
addition of increasing concentrations of the promoter oligonucleotides to a fixed concentration of 5Me.
More detailed investigations should be carried out to elucidate the mechanism of cell death in this regard.
TMPyP4 showed no specificity for tumor vs normal cells whereas 5Me showed significant specificity
which was reflected in MTT assay performed on PANC-1, B16F10, MDA MB 231 and CHO.
Earlier TMPyP4 biological effects were studied at cytotoxic concentrations for short term and at
subtoxic concentration for long term time duration.39 They reported that short term effects are associated
with telomere disruption, uncapping effects and down regulation of oncogenes. In this part of our study,
short term drug toxicity investigation is conducted and effects of 5Me for longer time duration is in
progress to prove its potentiality as anticancer agent. The phototoxicity of the two compounds is also
studied using Waldman 1200 light source and 5Me has shown more phototoxicity than TMPyP4 on
PANC-1 and SKOV3 cells and further studies to elucidate mechanism of cell death are under progress.
The huge difference in IC50 values of 5Me and 6Me might be due to more efficient uptake of 5Me
compared to TMPyP4 (6Me) after 2-3 h, at which time point cells were exposed to light. The triplet
quantum yield, singlet oxygen quantum yield etc also play important role in phototoxicity and further
such studies would establish 5Me as an efficient PDT agent.
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5. CONCLUSIONS
Porphyrins are known as anticancer agents due to their G4 DNA binding and photophysical properties.
TMPyP4 and a structurally modified derivative of TMPyP4 (5Me) are successfully synthesized and
characterized by MALDI-TOF and NMR. The photophysical investigations revealed that 5Me has
superior selectivity and stability towards G4 DNA over duplex DNA compared to TMPyP4. Cell culture
studies have shown that both are toxic to cancer cells but 5Me has shown lesser degree of toxicity to
normal cells compared to TMPyP4. Both the compounds are arresting the cell cycle at G2/M phase. 5Me
can exert its activity on cancer cell division at two regulatory levels, as it has binding affinity to
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telomeric G-quadruplex regions as well as to the promoter regions of oncogenes like c-myc, Bcl-2,
KRAS. The phototoxicity studies established that 5Me was more efficient in killing cancer cells than
TMPyP4. DNA binding studies using biophysical methods indicate that 5Me has very good interaction
with G-quadruplex DNA.
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Acknowledgements— we acknowledge DST for the financial support to Ushasri under WOS-A scheme
(SR/WOS-A/LS-1374/2014) (G) and LG thanks to DST (DST, SB/S1/IC-14/2014). We thank Dr. Radha
and Dr. Rakesh Mishra, CSIR-CCMB for allowing us to use Waldman light source. We greatly
acknowledge Dr. Arabinda Chaudhuri, Dr. RajKumar Banerjee, CSIR-IICT and Dr. Gopal Pande,
Manipal academy of higher education for their support.
Supporting Information: In the supporting information we provide MALDI-TOF and NMR spectra of
the compounds (Porphyrin 1, Porphyrin 2, 6Me and 5Me), ROS generation during treatment with
TMPyP4 (6Me) and 5Me studied by flow cytometry and UV-vis melting profiles of Tel Q, Tel M and
dsDNA and the respective complexes with 5Me and 6Me.
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