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Therapeutic potential of harmaline, a novel alkaloid, against cervical cancer cells in

vitro: Apoptotic induction and DNA interaction study

Article · May 2018

DOI: 10.7324/JABB.2018.60401

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Journal of Applied Biology & Biotechnology Vol. 6(04), pp. 1-8, July-August, 2018
Available online at http://www.jabonline.in
DOI: 10.7324/JABB.2018.60401

Therapeutic potential of harmaline, a novel alkaloid, against cervical

cancer cells in vitro: Apoptotic induction and DNA interaction study

Paromita Bhattacharjee, Sarita Sarkar, Tapas Ghosh, Kakali Bhadra*

Department of Zoology, University of Kalyani, Kalyani, Nadia, India.

ARTICLE INFO ABSTRACT

Article history:
The study emphasizes the growth inhibitory effect of harmaline on HeLa (human cervical cancer) cell line and mode
Received on: February 17, 2018
and mechanism of binding with CT DNA (calf thymus DNA). The results of cytotoxic study performed through MTT
Accepted on: March 14, 2018
assay indicates that harmaline have concentration dependent growth inhibitory effect on HeLa cell line with GI50
Available online: May 22, 2018
value of 28 µM. Furthermore, the alkaloid induced DNA damage and changes in mitochondrial membrane potential
in the cell line. The alkaloid shows reactive oxygen species dependent cellular damage with significant arrest in G2/M
Key words: Beta carboline,
population of the cell. Biophysical experiments further established the Kiw value (product of cooperative binding
Cytotoxicity, Reactive oxygen species,
Spectroscopy, Isothermal calorimeter. affinity and the cooperative factor) of harmaline with CT DNA to be 5.60 × 105 M−1. Harmaline showed a progressive
quenching of the fluorescence emission spectra. Circular dichroism study shows significant structural changes of
DNAwith subsequent induction of optical activity in the bound achiral alkaloid molecule. The alkaloid stabilizes
the DNA to 10°C. Intercalated state of harmaline inside DNA helix was shown by viscometric and ferrocyanide
quenching. The results highlight the importance of alkaloid- DNA interaction for developing nucleic acid based
therapeutic agents.

1. INTRODUCTION inhibition [15], and in vitro human topoisomerase I inhibition

effects [16] and in vitro cytotoxic effects [17]. Another beta carboline
Cervical cancer is the third most frequently occurring cancer, and
alkaloid harmine and its derivatives were also proved to be cytotoxic
the fourth leading cause of cancer mortality in women worldwide,
against different cancer cell lines [18].
with an estimation of 530,000 new cases and 175,000 mortality each
year [1]. Nucleic acid, chiefly DNA which is the main hereditary Previously, harmalol, another naturally occurring beta carboline
and genetic element of the cell, is the target biomolecule in the alkaloid, have been reported to have strong in-vitro cytotoxic
evolution of cancer. The use of alkaloids as medicine by humans date ability [19-21] and also have binding potential with nucleic
back thousands of years and the most important of them includes acids [22,23]. Hence, with reference to our previous investigation, in
morphine, quinine, taxol, camptothecin, and berberine. Besides other
this study we have tried to explore the binding and cytotoxic ability
pharmacological importance, plant alkaloids have been found to be
of harmaline on human cervical carcinoma (HeLa) cell line in detail
potent anti-cancer agent targeting deoxyribonucleic acids [2-6]. Beta
using different biophysical, calorimetric, and biochemical assays. This
carboline alkaloids are one of such plant alkaloids, which have proven
to be highly cytotoxic with several biological and nucleic acid binding research will help to design DNA based cancer therapeutic drug in the
effects [7-9]. Recently, they have also been proved to cause oxidative future.
modification of low-density lipoprotein particles, which can reduce
severe atherosclerosis [10]. Harmaline [Figure 1], a di-hydro beta 2. MATERIALS AND METHODS
carboline alkaloid, is such a plant alkaloid isolated from the seeds and
2.1. Materials
roots of Peganum harmala (Syrian rue) [11] and have wide range of
biological actions including anti-leishmanial [12], insecticidal [13], Harmaline hydrochloride was obtained from Sigma-Aldrich (St.
acetylcholinesterase inhibition [14], central monoamine oxidase Louis, MO, USA). Its concentration was determined using molar
extinction coefficient value of 18,527 M−1/cm at 371 nm. CT DNA was
also purchased from Sigma—Aldrich. Concentration was determined
*Corresponding Author:
Kakali Bhadra, Department of Zoology, University of Kalyani, Kalyani, using the molar extinction coefficient (ε) of 6600 M−1/cm at 260 nm as
Nadia,741235, India. described earlier [22,24,25]. Harmaline and CT DNA were dissolved
E-mail: [email protected] in 15 mM Citrate–Phosphate (CP) buffer of pH 6.8.

© 2018 Paromita Bhattacharjee, et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-
ShareAlike Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
2 Bhattacharjee, et al. Journal of Applied Biology & Biotechnology 2018;6(04):1-8

2.2. Cell Viability Test and Other Biochemical Assays and stained with propidium iodide (PI). The experimental protocol was
2.2.1. MTT assay followed an earlier report [20,26]. The histograms were obtained by
HeLa and WRL-68 cells were obtained from National Centre for BD Science FACS (Caliber), and the data were analyzed using the
Cell Science Pune. Cell growth protocols were followed as described Cyflogic software.
earlier. Cells were grown in DMEM (HiMedia) medium supplemented
2.2.5. Quantitative analysis of intracellular reactive oxygen
with 10% heat inactivated fetal bovine serum (HiMedia) and 1%
species (ROS) generation by flow cytometry
antibiotic antimycotic solution (HiMedia), at 37°C with 5% CO2. The
HeLa cells (2 × 105/35 mm culture petridish) were treated with
percentage of viable cells were calculated as the GI50 (50% growth
harmaline (28 µM) for 8 and 16 h and thereafter incubated with
inhibition) values for the cell lines by MTT (1 mg/mL of the MTT)
DCFH-DA (20 µM). Intracellular ROS was determined and quantified
assay kit (Sigma), by the following formula as reported earlier [20,26].
by fluorescence-activated cell sorter (BD Science Calibur).
GI50 = (T-TO) ×100/(C-TO)(1)

Where, T represents the optical density of the sample culture plate

2.3. Statistical Analysis
after 48 h of harmaline treatment, To is the optical density at time zero MTT assay data are statistically analyzed with ANOVA test, and
and C is the optical density of control. The average value of three significant values are calculated against both untreated as well
independent experiments was taken. as solvent control (P < 0.05 vs. solvent control). Every data is
representative of three independent experiment.
2.2.2. Lactate dehydrogenase (LDH) enzyme assay
LDH activity was measured for both untreated and treated cells by
2.4. Spectroscopic and Calorimetric Experiments
following the work done by Biswas et al. [27] using Coral, India, LDH
assay kit. HeLa cells (2 × 104 cells/35 mm culture plate) were treated We used Jasco V-630 spectrophotometer (Jasco International Co.
with various concentration of harmaline (13, 28 and 37 µM) at 37°C Ltd. Tokyo, Japan) to perform the absorbance measurements. After
for 48 h. Percent viable, apoptotic and necrotic cells were determined addition of the alkaloid to the CT DNA solution (40 µM) each time,
as our previous work [20]. from the absorbance at the isosbestic point at 393 nm for the CT DNA,
the total drug concentration (Ct) present was calculated as Ct = Aiso/
% apoptosis = (LDHp × 100%)/(LDHp + LDHi + LDHe)(2) emax. This quantity was used to calculate the expected absorbance (Aexp)
at wavelength maximum, Aexp = Ctemax. The difference in Aexp and the
% necrosis = (LDHe × 100%)/(LDHp + LDHi + LDHe)(3)
observed absorbance (Aobsd) was then used to find out the total amount
After collecting the suspended cells from culture media by centrifugation of bound harmaline as Cb = A/∆e = (Aexp-Aobsd)/(ef-eb). Free harmaline
(3000 r.p.m for 4 min at 4°C), the LDH content from the pellets was concentration was obtained by the difference, Cf = Ct-Cb. The molar
marked as apoptotic cells (LDHp). The extracellular LDH in the culture extinction coefficient of completely bound harmaline was determined
supernatant was marked as necrotic cells (LDHe), and the LDHi present by adding a known quantity of harmaline to a large amount of DNA
in the adherent viable cells was used as intracellular LDH. polymer and on the assumption of total binding, eb = Amax/Ct. This
binding data was plotted into Scatchard plots (r/Cf versus r where r is
2.2.3. Microscopic studies the number of moles of harmaline bound per mole of DNA nucleotide)
Phase contrast microscopy (carl zeiss microscope) was used for which was studied further for cooperative binding using the following
determining growth inhibition property of the alkaloid on untreated equation of McGhee and von Hippel (McGhee, and von Hippel, 1974).
and treated HeLa cell population.
(n −1) 2
Nuclear fragmentation and mitochondrial membrane potential changes r  (2ω+1)(1 − nr) + (r − R)   1 − (n + 1)r+R 
= K i (1 − nr) ×    
were performed using 4′,6′-diamidino-2-phenylindole (DAPI) (5 µM) Cf  2( ω − 1)(1 − nr)   2(1 − nr) 
and JC-1 (7 µM) dye obtained from Sigma respectively, in control and (4)
treated cells (2 × 104 cells/mm in 35 mm culture plate) by OLYMPUS
fluorescence microscope. Where, R = {[1-(n+1)r]2+4ωr(1-nr)}1/2, Ki is the intrinsic binding
constant to an isolated binding site, n is the number of base pairs
Comet assay (single cell gel electrophoresis) was performed on excluded by the binding of a single drug molecule and ω is the
single HeLa cells to find out nuclear fragmentation which is again an cooperative factor. The binding data were analyzed using Origin 7.0
important apoptosis indicator [20,28]. 0.5 mg/mL ethidium bromide (Origin Lab Corporation, Southampton, MA, USA) software.
solution of was added to the gel and the stained DNA in the cells
was examined at ×200 magnification with the help of OLYMPUS DNA Melting data were recorded on a Jasco V-630 unit associated
Fluorescence Microscope. The percentage of the DNA damage was with a Peltier controlled Jasco PAC-743 model accessory (Jasco
measured using the software CASP. International Co. Ltd. Tokyo, Japan). The experimental protocols are
same as described previously [22].
Besides that, after fixing the cells (2 × 104 cells/35 mm culture plate)
with 4% paraformaldehyde (Sigma) solution, they were cleaned by Fluorescence spectra were obtained through a Hitachi F4010
tetrachloromethane, air-dried and the gold coating was performed by fluorescence spectrometer (Hitachi Ltd., Tokyo, Japan) in fluorescence
IB2 ION COATER. Then, three dimensional morphology of control free quartz cells having 1 cm path length. The excitation wavelength
and treated (with 28 µM harmaline) cells was studied by Cambridge for the drug was 376 nm, and the experiment was performed at 25 ±
250 scanning electron microscope (SEM). 0.5°C. The excitation and emission band passes were 2.5 and 10 nm,
respectively. Quantum yield calculations were performed following
2.2.4. Cell cycle arrest study the equation of Parker and Rees (1960) as described earlier [25],
HeLa cells after growing at a density of 2 × 105 cells/mL in 35 mm
culture plate were treated with 13, 28 and 37 µM of harmaline for 48 h fs = (FseqCq × 0.55)/(Fq. esCs)(5)
 Bhattacharjee, et al.: Harmaline inhibiting HeLa cells in vitro 2018;6(04):1-8 3

Where, F is the integrated area of the fluorescence emission curve in 50 µM. These findings clearly indicated that the alkaloid has a strong
an arbitrary unit, e represents the molar extinction coefficient and C growth inhibition property on epithelial cancer cells in comparison to
represents the molar concentration of sample (s) and quinine sulfate the normal epithelial cell line. To further strengthen the finding, LDH
(q), respectively. Quinine sulfate in 0.1N H2SO4 was utilized as assay was performed. This assay resulted in 4.7 ± 1.1% necrotic, 58.5%
reference standard for quantum yield measurements. ± 1.2% apoptotic and 36.8 ± 0.90% viability in harmaline treated cell
line, whereas, in control cell line the values were 4.8 ± 1.2%, 11.2 ±
CD spectra were measured on a Jasco J815 spectropolarimeter 0.9%, and 84.0 ± 2.1%, respectively [Figure 2b].
attached to a temperature controller, temperature programmer (model
PFD 425 L/15) and a PC, as described previously [21,22,23,29]. Nucleic acid-specific fluorochromes emit different fluorescent color
when bound to target nucleic acids and this event can be observed
GE Microcal Isothermal calorimeter (ITC) 200 (Northampton, USA) under fluorescence microscopy [20]. Cellular morphological changes
was used for ITC experiment, and Origin 7.0 software was used for such as rounding of cell contour and fragmentation of nucleus were
the analysis. determined by phase contrast microscopic images [Figure 2c].
With increasing drug treatment, HeLa cells revealed increasing
2.5. Analysis of Mode of Binding oligonucleosomal fragmentation and nuclear condensation after
Using anionic quencher [Fe(CN)6]4_, fluorescence quenching study treatment with DAPI [Figure 2d]. Comet assay was performed
was performed as described previously [28,29]. The data were plotted thereafter, that showed DNA strand breaks in the comet tail constituting
as Stern–Volmer plots of Io/I versus [Fe (CN)6]4_. of 24 ± 1.0% amount of DNA after GI50 dose (28 µM) treatment and
the values were calculated by software CASP [Figure 2e and f]. We
A Cannon-Manning Type 75 semi-micro viscometer was used. Linear further performed mitochondrial membrane potential assay using JC-
sonicated CT DNA was used as the sample, as described earlier [25]. 1, a voltage sensitive fluorescent cationic dye, which accumulates in
Relative viscosities for DNA, both in the presence or absence of mitochondria after sensitizing different voltage and indicates decrease
harmaline was calculated from the relation. in membrane potential by a fluorescence emission change from red to
green [Figure 2g]. Morphologic description of apoptosis using SEM
 t complex − t ′o  still remains one of the best ways to define apoptosis as apoptotic
 
ηsp  t ′o  cells contain many morphological alterations than control cells [20].
= To validate our previous results, the three-dimensional morphological
η sp  t control − t o  (6)
  changes in control and harmaline treated cells were observed by
 to  SEM [Figure 2h]. While, untreated HeLa cells have shown the
typical morphological features of cervical cancer cells which include
Where, h¢sp and hsp are specific viscosities of the alkaloid-nucleic acid cell surface projections, several morphological alterations (namely
complex and the nucleic acid respectively, tcomplex, tcontrol, t¢o and to are membrane blebbing, cell shrinkage etc) were observed in a large
the average flow times for the complex, free nucleic acid, solvent for number of treated cells.
the complex and solvent for the free nucleic acid respectively.
PI staining for cell cycle analysis indicates aggregation of cell
3. RESULTS AND DISCUSSIONS population at G2/M stage, the detailed result is depicted in Table 1
[Figure 3a and b]. ROS generation is a key role of many anticancer
compounds [32]. Furthermore, cancer is a probable cause of various
3.1. Cell Viability Study and Other Biochemical Assays
chemical induced ROS dependent stresses in the body [33]. Maximum
Growth inhibition capacity of Harmaline against one of the most of the chemotherapeutic agents inhibit cancer cells growth by
prevalent human cervical cancer cell lines, HeLa and normal epithelial preventing intracellular ROS elevation [33]. In this study, harmaline
cell line, WRL-68 was studied by MTT assay. HeLa and WRL- has been shown to cause ROS mediated apoptosis in the cancer cell
68 cells were treated with different concentrations of harmaline (10, line with a subsequent cell cycle arrest at G2/M phase, thereby acting
20, 30, 40, and 50 µM) at 37°C for 48 h [Figure 2a]. It was found as a chemotherapeutic agent. The effects of harmaline on HeLa cells
that harmaline has the GI25, GI50 and GI75 values of 13 µM, 28 µM were found to be time-correlated as observed by the flow cytometric
and 37 µM, respectively, in HeLa cells; while no prominent growth quantification of ROS generation study after adding DCFH-DA in
inhibition was found in WRL-68 cells after harmaline treatment till harmaline treated (28 µM) cells for 8 and 16 h [Figure 3c]. It was
measured using excitation/emission wavelengths of 500/520 nm as
reported earlier [20].

As the alkaloid shows nuclear fragmentation targeting DNA, several

spectroscopic and calorimetric analyses were performed to emphasize
the mode and mechanism of binding with CT DNA.

3.2. Spectroscopic and Calorimetric Studies

The characteristic ultraviolet (UV)- visible spectrum of harmaline (red
color) at pH 6.8 exhibits maxima at 205, 258, and 371 nm, respectively,
in the range of 200–550 nm. With increasing concentration of CT DNA,
characteristic hypochromic (27.4%) effect and bathochromic shift
(7 nm) was observed [Figure 4a]. The hypochromic effect essentially
indicates strong intermolecular interaction involving effective overlap
Figure 1: Chemical structure of harmaline. of the π electron cloud of harmaline with the nucleotide bases. The
4 Bhattacharjee, et al. Journal of Applied Biology & Biotechnology 2018;6(04):1-8

spectra show prominent isosbestic point at 393 nm, indicating and found to be a non-linear curve [Figure 4a]. In the case of CT DNA
equilibrium. The findings from UV spectroscopic absorption titration with harmaline, where a positive slope was observed at low values of
of increasing concentration of harmaline to a fixed concentration of bound alkaloid, the analysis for cooperative binding was performed
CT DNA was expressed in the form of Scatchard plot (r/Cf versus r) using the McGhee–von Hippel Eq. (4). Generally, cooperative binding

Table 1: Percentage of HeLa cell population at the various cell cycle stagesa.
Drug concentration (µM) Sub G0/G1 cell population (%) G0/G1 cell S cell G2/M cell
population (%) population (%) population (%)
Control 0.82±0.3 85.08±1.03 3.31±0.5 10.79±0.5
13 1.57±0.3 66.61±1.0 12.39±0.9 19.43±0.9
28 8.67±0.5 40.98±0.5 16.44±1.0 33.91±0.7
37 8.72±0.9 36.67±0.3 17.45±1.0 37.16±1.0
aAverage of three experiments are presented here and the means are significant compared with their control (P<0.05).

a b

c
h

e f

g
Figure 2: (a) MTT assay on HeLa and WRL-68 cell lines after harmaline treatment of various concentrations (13, 20, 30, and 40 µM) for 48 h. Data are
statistically analyzed with ANOVA test and significant values are calculated against both untreated as well as solvent control (*P < 0.05 vs. solvent control),
(b). LDH assay performed after harmaline treatment to find out percentage of apoptotic and necrotic cell death. Data represent three independent experiments,
(c). Phase contrast microscopic images (×200) of control and treated HeLa cells, (d). Nuclear fragmentation of HeLa cells induced by 28µM and 37µM harmaline
in comparison to control cells after staining the nuclei with DAPI and observed under a fluorescence microscope (×200), (e). Comet assay showing prominent
comet tails harmaline treatment indicative of DNA damage, (f). Comet assay bar graph representing % DNA damage in HeLa cells after drug treatment (GI50) by
software CASP. The graph represents the mean ± standard deviation of three independent experiments, (g). HeLa cells were treated with and then subjected to
JC-1 staining to evaluate the changes in mitochondrial membrane potential viewed under fluorescence microscope (×200), (h). Changes in cell shape and contour
after 28 µM harmaline treatment as observed under S530 Hitachi scanning electron microscope.
 Bhattacharjee, et al.: Harmaline inhibiting HeLa cells in vitro 2018;6(04):1-8 5

is mediated by allosteric effects [21,24,25,29-31]. Scatchard plot 105/M with a stoichiometry of 4 nucleotide phosphates. The binding
from the interaction of harmaline with CT DNA indicates [inset of constant value was quiet close to the previous result by Nafisi et al.,
Figure 4a] intrinsic binding affinity, Kiω (product of cooperative 2010 [9]. Stabilization of the alkaloid to CT DNA was also studied
binding affinity and the cooperative factor) in the order of 5.60 × by optical melting experiment, which revealed that the native melting

a c
Figure 3: (a). Histrogram of HeLa (Control and treated with 13, 28 and 37 µM of harmaline), obtained from FACS analysis after staining with propidium iodide.
(b). Bar graph represents the values of cell count in G0/G1 and G2/M phase for control and treated cells with increasing concentrations (13, 28 and 37 µM) of
harmaline. Three independent experiments were performed and the means are significant in comparison with their control (P < 0.05). Complete data is presented
in Table 1, (c). Harmaline induced ROS generation in HeLa cells. HeLa cells were cultured in absence (control, green) and presence of harmaline for the specified
time period. DCFDH-DA fluorescence intensity was detected by using flow cytometry.

a b

c d
Figure 4: (a). Absorption spectra of harmaline (10 µM) in 15 mM CP buffer, pH 6.8 at 25 ± 0.5°C (red spectra) and with treatment of 53.45 µM CT DNA (black
spectra); (insight) Scatchard plots derived from absorbance spectral data for the binding of harmaline to 20 µM of CT DNA. The data was fit to the cooperative
binding model derived from McGhee-von Hippel equation, (b). Steady state fluorescence emission spectrum of harmaline (3 µM, red colour spectra) to 7.20,
15.60, 28.90, 34.40, 48.40, 54.30, 63.10 and 72.6 µM (curves 2–8) of concentrations CT DNA µM of CT DNA and plots (Inset) of the relative quantum yield f/f
versus P/D for the interaction of harmaline (■-■) with CT DNA, (c). CD spectra resulting from the interaction of harmaline with CT DNA (65 µM) in 15 mM CP
buffer of pH 6.8 at 25 ± 0.5°C. Curves 1–7 denote CT DNA (65 µM) treated with 0, 5.0, 12.0, 15.0, 36.5, 48.7 and 55.0 µM of harmaline (d). Extrinsic CD spectra
of CT-DNA and harmaline interaction.
6 Bhattacharjee, et al. Journal of Applied Biology & Biotechnology 2018;6(04):1-8

temperature of CT DNA (67°C) at 15 mM is stabilized to 10°C in the 242 nm negative peak show only slight changes in molar ellipticity.
presence of alkaloid [Figure not shown]. A prominent extrinsic CD peak in the region of 376 nm [Figure 4d]
was also obtained.
Fluorescence emission spectra of harmaline-DNA interaction were
recorded in 400–650 nm [Figure 4b]. The alkaloid was found to A thermodynamic study through ITC was performed which is a direct
be a strong fluorophore having emission spectral peak at 480 nm methodology of interpreting various thermodynamic parameters in
when exited at 376 nm and showed 56% quenching of steady state various bio molecular interaction studies [37-39]. A reversed protocol was
fluorescence intensity during the interaction. The quantum yield (f/fo) used throughout the experiment as the alkaloid has high heat of dilution
of the alkaloid-DNA complexes [Table 2] depicts a value of 0.29 at [Figure 5]. Previously, Yang et al. and Du et al. (2004) had reported the
saturation P/D 24.2 [inset of Figure 4b]. thermodynamic parameters for the interaction of beta carboline derivatives
with DNAs [40,41]. The thermodynamic data are depicted in Table 2 and
Conformational changes of CT DNA- harmaline was studied by the binding constant (Kb) and stoitiometry (N) of 5.60 ± 0.02 × 105/M
circular dichroism (CD) [21,23,34-36] [Figure 4c]. The intrinsic CD and 4.0, respectively, have been found which are in good agreement with
spectra of the CT DNA-alkaloid interaction at pH 6.8 shows a regular the spectrophotometric data. Furthermore, the large negative enthalpy
and prominent perturbation in the 278 nm positive peak while the change of −5.10 ± 0.02 kcal/mol has been reported which is typical case
for intercalative binding of the drug -DNA interaction [42].

3.3. Mode of Binding

The binding mode of harmaline to the sonicated CT DNA has been
studied by viscosity experiments [Figure 6a]. As the D/P (alkaloid/
nucleotide phosphate) increase, the relative specific viscosity of
CT DNA–harmaline complex increased sharply, suggesting the
intercalation of the alkaloid into the DNA helix. The changes were
due to the increase in the axial length of the DNA and it becomes more
rigid after binding with the alkaloid. Hence, the viscosity increases as
both the factors enhanced the frictional coefficient. The above changes
were compared with CT DNA-ethidium bromide complexation as
ethidium is used as a classical intercalator.

Ferrocyanide ([Fe(CN)6]4−) quenching is another reliable method for

studying the mode of binding of ligands to nucleic acids. The study shows
a Ksv value of 177 ± 1.2 M−1 for free harmaline with ferrocyanide. While
Figure 5: Isothermal calorimeter profile for binding of 1200 µM of CT DNA that of bound molecule to CT DNA was 56.7 ± 0.7 M−1 indicating that the
to harmaline (22 µM) at 25 ± 0.5°C, pH 6.8. Each heat burst curve (in the bound alkaloid was considerably sheltered and sequestered away from the
bottom part of upper panel) is due to the 1.5 µL sequential injection of the solvent suggesting good stacking inside the DNA helix [Figure 6b].
DNA into the alkaloid (curves at the bottom). The data points (■-■) reflect the
4. CONCLUSION
experimental injection heats while the solid line represents the calculated best
fit of the data. The values of the various thermodynamic parameters obtained For the development of a drug it is an absolute necessity to understand
are presented in Table 2. the molecular interaction of the drug and its cellular target molecules.

a b
Figure 6: (a). Plot of changes of relative specific viscosity of sonicated (●-●) CT DNA with increasing concentration of harmaline. The relative specific viscosity
of the polymer was compared with the change in specific viscosity of CT DNA with ethidium (▲-▲). The specific viscosity was calculated from equation (6)
described in text. The concentration of DNA was 450 mM, respectively, (b). Stern-Volmer plots for quenching by increasing concentration of [Fe(CN)6]−4 with
harmaline in the absence of polymer (●-●) and in the presence of (■-■) CT DNA. The corresponding K’sv values are presented in Table 2.
 Bhattacharjee, et al.: Harmaline inhibiting HeLa cells in vitro 2018;6(04):1-8 7

Results of our study revealed that harmaline have potent cytotoxic

Data presented from the average of four determinations in each. φ/fo denotes the relative quantum yield value at saturation of nucleotide/alkaloid molar ratio determined as described in the text. bAverage of three determinations in CP buffer,
(kcal/mol)
property by inducing ROS dependent apoptosis in the human cervical

ΤDS°

2.83
cancer cell line, HeLa and furthermore, shows conformational changes
in DNA. Hence, this research work is providing sufficient informations
to conclude harmaline as a potent chemotherapeutic drug against
human cervical cancer and will be developed in future as a better and

−5.10±0.02
(kcal/mol)
Calorimetry (ITC) b effective beta carboline derivative for biomedical uses.
∆Ho
Table 2: Quantitative data for the interaction of harmaline with CT DNA in 15 mM CP buffer at 25±0.5°C obtained from spectrophotometrica, spectrofluorimetrica and calorimetric studiesb

5. ACKNOWLEDGMENTS

PB has been supported by grant from University of Kalyani. SS and

−7.93±0.03
(kcal/mol)

TG are supported by DST, WB and UGC respectively. Authors are

∆Go

thankful to DST-RFBR 2017-19 (DST/INT/RUS/RFBR/P-254)

and PRG-2017-18, University of Kalyani and to the Department
of Environmental Science, University of Kalyani for providing
spectrofluorimeter and fluorescence microscope facility.
6.05±0.12
(×105/M)
Kb

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