Novel flourescent spiroborate esters: potential therapeutic agents in in vitro cancer models

Article abstract of DOI:10.1007/s11033-018-4529-5

The current treatment system in cancer therapy, which includes chemotherapy/radiotherapy is expensive and often deleterious to surrounding healthy tissue. Presently, several medicinal plants and their constituents are in use to manage the development and progression of these diseases.They have been found effective, safe, and less expensive. In the present study, we are proposing the utility of a new class of curcumin derivative, Rubrocurcumin, the spiroborate ester of curcumin with boric acid and oxalic acid (1:1:1), which have enhanced biostability for therapeutic applications. In vitro cytocompatibility of this drug complex was analysed using MTT assay, neutral red assay, lactate dehydrogenase assay in 3T3L1 adipocytes. Anti tumour activity of this drug complex on MCF7 and A431 human cancer cell line was studied by morphological analysis using phase contrast microscopy, Hoechst staining and cell cycle analysis by FACS. To explore the chemotherapeutic effect, the cytotoxic effect of this compound was also carried out. Rubrocurcumin is more biostable than natural curcumin in physiological medium. Our results prove that this curcumin derivative drug complex possess more efficacy and anti-cancer activity compared with curcumin. The findings out of this study suggests this novel compound as potential candidate for site targeted drug delivery.

Full text of DOI:10.1007/s11033-018-4529-5

Molecular Biology Reports
https://doi.org/10.1007/s11033-018-4529-5
ORIGINAL ARTICLE
Novel flourescent spiroborate esters: potential therapeutic agents
in in vitro cancer models
S. Anjana1 · Josna Joseph2 · Jeena John3 · S. Balachandran3 · T. R. Santhosh Kumar4 · Annie Abraham2
Received: 1 September 2018 / Accepted: 26 November 2018
©
Springer Nature B.V. 2018
Abstract
The current treatment system in cancer therapy, which includes chemotherapy/radiotherapy is expensive and often deleteri-
ous to surrounding healthy tissue. Presently, several medicinal plants and their constituents are in use to manage the devel-
opment and progression of these diseases.They have been found effective, safe, and less expensive. In the present study,
we are proposing the utility of a new class of curcumin derivative, Rubrocurcumin, the spiroborate ester of curcumin with
boric acid and oxalic acid (1:1:1), which have enhanced biostability for therapeutic applications. In vitro cytocompatibility
of this drug complex was analysed using MTT assay, neutral red assay, lactate dehydrogenase assay in 3T3L1 adipocytes.
Anti tumour activity of this drug complex on MCF7 and A431 human cancer cell line was studied by morphological analy-
sis using phase contrast microscopy, Hoechst staining and cell cycle analysis by FACS. To explore the chemotherapeutic
effect, the cytotoxic effect of this compound was also carried out. Rubrocurcumin is more biostable than natural curcumin in
physiological medium. Our results prove that this curcumin derivative drug complex possess more efficacy and anti-cancer
activity compared with curcumin. The findings out of this study suggests this novel compound as potential candidate for
site targeted drug delivery.
Keywords Curcumin · Skin cancer · Drug delivery · Natural product · Anti-cancer agent
Introduction
result due to inflammation [2] necessitating the search for a
drug with anti-inflammatory property with low side effects.
Though the cancer of colon, lung, prostate and breast
occur commonly around the world, there are only ten times
lower incident reports in India [3]. This may be attributed to
the regular consumption of the flavonoid curcumin contain-
ing turmeric, which is a prominent spice in Indian cuisine.
Curcumin plays an important role in slowing down the pro-
gression of skin cancer and it has curative properties. It is
hypothesized that curcumin has growth-inhibitory effects
through the TOR pathway [4] and chemopreventive poten-
tial in skin sarcoma where local application could bypass
bioavailability problems. It has also been reported that cur-
cumin had been used in the form of a skin ointment for the
treatment of basal cell carcinoma [5].
Skin cancer cases are widely being reported worldwide. It is
estimated that more than 9500 people in the U.S. are diag-
nosed with skin cancer every day [1], whereas fewer inci-
dence are reported in tropical nations like India and Africa.
The major types of skin cancer are basal cell carcinoma,
squamous cell carcinoma and melanoma. The current treat-
ment regimen including surgery, radiotherapy and chemo-
therapy cream are effective in managing the situation, but
with their own side effects. Also, skin cancer can at times
*
Annie Abraham
annieab2013@gmail.com
1
2
3
4
Department of Biotechnology, University of Kerala,
Thiruvananthapuram, Kerala, India
Of all types of skin cancers, melanoma is the most fatal.
Curcumin arise as a potent natural drug,which is powerful
enough to arrest the growth of cancer cells without being
cytotoxic to normal cells [6]. Sometimes, melanoma can
become resistant to chemotherapy and in such cases turmeric
offers greater application. This is because curcumin is able
to trigger the clean up of dysfunctional cellular components
Department of Biochemistry, University of Kerala,
Thiruvananthapuram, Kerala, India
Department of Chemistry, MG College,
Thiruvananthapuram, Kerala, India
Rajiv Gandhi Centre for Biotechnology,
Thiruvananthapuram, Kerala, India
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[
7]. Curcumin could prevent skin cancer and it protects the
ointments contain oxalic acid and boric acid, which are well
known antibacterial agents. Reports show that combina-
tion of oxalic acid, boric acid and curcumin could result in
the formation of another compound, rubrocurmin which is
highly stable in oil medium [10].
skin by eliminating free radicals and reducing inflammation
through inhibition of factors involved in tumour formation.
Besides, it also protects the skin from various kinds of oxi-
dative stress [8]. This is attributed to triggering phase II
detoxification enzymes which have prominent role in detoxi-
fication reactions.
In the present study, a series of mixed spiroborate esters
of curcumin were subjected to preliminary analysis against
different cancer cells. Four spiroborate esters of curcumin
formed by the condensation of oxalic acid, malonic acid,
salicylic acid and citric acid with curcumin and boric acid
(Fig. 1) were used for the primary analysis against cancer
cells. Among these, the oxalic acid complex, Rubrocurcumin
is a suggestive remedy as a drug releasing agent, since it is
highly stable in organic medium and it slowly decomposes
to curcumin in aqueous organic medium. Siu et al. observed
that Rubrocurcumin is active against HIV protease and its
hydrolyzed product is a well-established biological active
reagent [14]. To the best of our knowledge, this is the first
study evaluating the metal complex of naturally occurring
bioactive food compound, curcumin on skin cancer cells.
Due to the special structure of this compound, an increase
in bioavailability and biostability is attributed. The findings
of this study would pave newer ways for site directed cancer
therapy, by suggesting Rubrocurcumin as a suitable drug
candidate.
Low bioavailability of curcumin is an impediment to its
transition to clinical trials. Application of curcumin con-
taining cream for skin cancer treatment, solves this problem
since curcumin is known to slow down the progress of the
skin cancer. In spite of its promising chemotherapeutic activ-
ity, pre-clinical and clinical studies reveal curcumin’s limited
therapeutic application, due to its instability in physiological
condition, poor solubility and higher metabolic activity [9].
One of the highly promising approaches to overcome this
instability and bioavailability issue is to convert curcumin
to its metal complex and many such curcumin complexes are
reported in literature [10]. Spiroborate esters of curcumin are
of good choice since they are more stable than curcumin in
solid state and in water mediated condition [11, 12]. More
over, the components of these complexes i.e., curcumin,
boric acid and dicarboxylic acid/hydroxy acids are in use
in many ayurvedic and other medical formulations [13] and
are reported to be non toxic. Majority of skin protection
Fig. 1 The chemical structure of synthesised spiroborate complex of Curcumin with oxalic acid (CBO), Malonic acid (CBM), salicylic acid
CBS) , Citric acid (CBC)
(
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Materials and methods
Cellular uptake studies
All chemicals and reagents used were of analytical grade.
All biochemical kits were purchased from M/s Sigma,
USA. The cell lines used in the study A431, (skin cancer
cells) were procured from NCCS Pune, MCF-7 (breast
cancer cells) from RGCB, Poojapura and normal cell
The internalization of drug complex (Rubrocurcumin,
CBO) was examined in A431 cells by using fluorescence
microscopy. Drug has a fluorescent property, so we used
this drug for cell imaging and further checked the cel-
lular uptake of this material. Cells were seeded at a den-
4
(
3T3L1) was procured from Department of Biotechnol-
sity of 5 × 10 cells/mL and allowed to incubate at 37 °C
ogy, University of Kerala. The ethical approval for this
study has been sought from the Institute Animal Ethics
Committee (IAEC), University of Kerala, sanction No.
IAEC-KU-1/2014-15-BC-AA (38).
for 24 h in culture medium supplemented with 10% FBS.
After incubation the cells were treated with drug for 24 h,
washed with PBS and further analysed the cellular uptake.
Cytotoxicity assays
The synthesis of spiroborate esters
Cytotoxicity in 3T3L1 adipocytes and MCF7, A431 cancer
cells
Curcumin which was separated from commercial sample
of curcuminoids using silica gel column chromatogra-
phy [15] is used for the synthesis of spiroborate esters
of curcumin. A mixture of curcumin (0.01 M), boric acid
Toxicity of the drug complex (Rubrocurcumin, CBO) to
a normal cell line, 3T3L1 adipocytes and cancer lines,
MCF7, A431 was studied by culturing the cells with dif-
ferent concentration of the drug for different time inter-
vals. The assays used to determine cytotoxicity were MTT,
NRU and LDH leakage assays. Also nuclear condensation
studies by Hoechst staining ewas carried out. Qualitative
evaluation of cytotoxicity like observation of changes in
cell morphology and adherence was also noted.
(
0.01 M) and the dicarboxylic acid (oxalic acid, malonic
acid)/hydroxy acid (salicylic acid, citric acid) (0.01 M)
in 10 mL toluene were stirred for 3–5 h at 90–100 °C.
The formation of complex was monitored through Thin
Layer Chromatography (TLC) and after the completion
of the reaction the solvent was removed by filtration to get
the solid product. It was further washed with toluene to
remove unreacted curcumin and recrystallized from ace-
tone. The organic structure of the synthesised molecules
is depicted in Fig. 1.
MTT cell proliferation assay [16] Cells were seeded in to
9
6 well microtetraplate (5000 cells/well) in 10% DMEM
and incubated for 24 h. Once the cells have attached,
drug was added in different concentration (100–300 µg/
ml) in duplicate and incubated for different time intervals
Stability and drug selection studies
The stability and degradation of the synthesised drug
complexes, spiroborate complex of curcumin with (A)
salicylic acid (CBS), (B) citric acid (CBC), (C) malonic
acid (CBM), oxalic acid (CBO) were studied in acqueous
medium like Dulbecco’s Modified Eagle’s Medium for dif-
ferent time intervals (2 h, 3 h, 4 h, 24 h) and the absorption
spectra was determined using UV–Vis spectrophotometer.
Also the morphological changes in cancer cell line upon
addition of drug (300 µg) was noted for selection of the
most anti-cancer effect for further in vitro studies.
(
24, 48, 72 h) at 37 °C. Cells treated with 10 mM con-
centration of cisplatin were taken as positive control and
another set maintained without any treatment as control.
After incubation, at each time interval the medium was
removed and equal volumes of fresh medium were added
along with 20 ml MTT (5 mg/ml) to each well. The plates
were kept for 3 h incubation. The yellowish MTT was
reduced to dark coloured formazan by viable cells only.
The formazan crystals formed were solubilized with MTT
lysis buffer (20% SDS in 50% dimethyl formamide). The
plate was kept protected from light, overnight at 37 °C in
an incubator. The color developed was quantitated with
ELISA plate reader (BioRad systems, USA) measuring at
wavelength 570 nm.
Spectroflurimetric analysis of compound
The fluorescent property of the selected drug complex
(
Rubrocurcumin, CBO) was checked by measuring the
The cells survival (CS) expressed as percentage was cal-
culated as follows.
emission spectra by a spectroflurimeter.
CS = (OD drug exposed cell/mean OD control wells) × 100
The graph was plotted by taking percentage viability in the
y-axis and concentration of drug in the x-axis.
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Neutral red uptake (NRU) assay [17] Cells were treated with
different concentration of drug (100–300 µg/ml) in duplicate
and incubated for different time intervals (24, 48, 72 h) at
tion of specific drugs and kept for incubation at 37 °C
for 24 h. After incubation the cells were taken from CO
2
incubator and medium was discarded and diluted dye was
3
7 °C. After incubation, a solution of neutral red, a vital dye
added to the wells. The cells were then incubated at 37 °C
was added to the 96 well plates. The plates were incubated
at standard culture conditions to allow neutral red uptake by
the cells. After 2 h incubation, decanted excess neutral red
and PBS was added to the wells. The solvent extracts the
neutral red dye contained within the cells. The plates were
placed on a plate shaker to fully extract the neutral red and
evenly distribute the dye in each well and absorbance with a
for 15 min in CO incubator and were observed under the
2
microscope.
Cell cycle analysis by FACS
The most commonly used dye for DNA content for cell
cycle analysis is propidium iodide. Propidium iodide is
a red colour stain which is used for cell cycle analysis to
determine in which is used for cell cycle analysis to deter-
mine in which phase of the cell cycle, the arrest is occur-
ring. It intercalates with major groove of DNA strand and
produces highly fluorescent adduct which can be excited at
488 nm with a broad emission entered on 600 nm, Since PI
can also intercalate with RNA, it is necessary to treat the
cells with RNase for optimal DNA resolution. For FACS
analysis, the cells were seeded in 12 well plates and the
drugs were added to the respective wells having sufficient
cell density. After 24 h of drug treatment the cells were
harvested with trypsin and collected in FACS tubes. These
tubes were centrifuged at 3500 rpm at 4 °C for 7 min, the
supernatant was discarded and the pellet was resuspended
in 1 ml of ice cold 1× PBS. Then the tubes were again
centrifuged at 3500 rpm at 4 °C for 7 min. The pellet was
dissolved in 0.3 ml of ice clod 1× PBS, this was followed
by the addition of 0.7 ml cold 70% ethanol, ethanol was
added drop wise while vortexing gently. Then kept the
tubes for 45 min, again centrifuged the tubes at 3500 rpm
at 4 °C for 7 min. Then the pellet was resuspended in
0.25 ml 1× PBS this was followed by the addition of 5 µl
of RNase into each tube, the tubes were incubated 37 °C
for 30 min. Then added 5 µl of PI stain into each tube, the
tubes were kept in dark for 5 min. Using 0.75 µl filter, the
suspension was filtered after diluting with 1× PBS, the
tubes were then loaded for analysis in a flowcytometer.
5
40 nm was measured using a micro plate reader (BioRad,
USA). The absorbance value (optical density) are then used
to determine the viability of each well comparing the opti-
cal density of the each material treated well compared the
negative control well.
LDH cytotoxicity assay [18] This is a colorimetric assay that
quantitatively measures LDH, a stable cytosolic enzyme
that is released into the culture medium upon cell damage or
lysis occurs during both apoptosis and necrosis [18]. LDH
catalyzes the reduction of NAD+ to NADH and H+ by
oxidation of lactate to pyruvate, which in turn catalyze the
reduction of a tetrazolium salt to a colored formazan and the
absorbance of the formazan developed can read at 490 nm.
The amount of formazan produced is proportional to the
amount of LDH released into the culture medium.
Cells were added in 24 well plates, exposed to drug
conjugated gold nanoparticle with different concentration
(
(
100–300 µg/ml) and incubated for different time intervals
24, 48, 72 h). After incubation the cell suspension was cen-
trifuged at 4000 rpm for 5 min and transferred to 100 ml of
the supernatant to each well in a microplate. Then added
1
00 µl of reaction solution to each well and incubated plates
with gentle shaking for 30 min at room temperature. Read
the absorbance at 490 nm using a plate reader (BioRad,
USA).
ꢀ
+ Alysed
ꢁ × 100
Atest = A
control
LDH activity can be determined by plotting a graph
against absorbance in y axis and time interval in x axis.
Results
Nuclear staining analysis—Hoechst staining Chromatin
condensation is a late apoptotic event, which can be deter-
mined by Hoechst staining. Hoechst 33342 is a DNA bind-
ing, non-intercalating, benzimidazole derivative dye that
binds differentially to A-T rich regions of DNA. This dye
is excited by UV light at 395 nm and emits blue fluores-
cence at 450 nm. Transport of Hoechst across the plasma
membrane is altered in apoptotic cells. Dye is diluted in
the buffer in the ratio 1:1000. The cells were grown in 96
well plates and apoptosis was induced in them by addi-
Synthesis and stability of spiroborane complexes
The synthesis and spectroscopic identification of spirobo-
rate ester formed from oxalic acid (CBO) and salicylic acid
(CBS) were reported recently elsewhere [11, 17]. The struc-
ture of CBM was confirmed by the spectral data which cor-
responds to that reported by Sui et al. [14]. The spectral
data of CBS correspnds to the methyl derivative of C was
reported earlier [19].
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Spectral details of CBM and CBS
CBM
116.03, 117.30, 125.73, 126.01, 148.21, 148.27, 151.86,
65.55, 177.58.
1
CBS
Yield 80%; UV λ = 516 nm (acetonitrile); IR (KBr):
max
3
512 (OH), 1670 (C=O in lactone ring), 1513 (C=O in cur-
Yield 75%; UV λ =500 nm (acetonitrile); IR (KBr): 3468
max
−
1
cumin), 1207(C–O in phenol),1058 cm (C–O in OCH );
(OH), 2888 (C–H), 1725 (C=O in lactone ring), 1553 (C=O
3
1
−1
H NMR (400 MHz, DMSO-d6); δ 3.84 (s, 6H, OCH ), 3.70
in curcumin), 1289 (C–O in phenol), 1081 cm (C–O in
3
1
(
s, 2H,CH2) 6.48 (s, 1H, CH), 7.36 (d, J=10 Hz, 2H, Ar–H),
OCH ); H NMR (400 MHz, DMSO-d6); δ 2.73–2.83 (m,
3
6
.89 (d, J=8 Hz, 2H, Ar–H), 7.45 (s, 2H, Ar–H), 8.02 (d,
J = 15.6 Hz, 2H, =CH), 7.07 (d, J = 15.6 Hz, 2H, =CH),
0.175 (s, 2H, OH); 13C NMR: 55.76, 100.47, 112.59,
4H, CH ), 3.8 (s, 6H, OCH ), 6.43 (s, 1H, CH), 7.32 (d,
2
3
J=10 Hz, 2H, Ar–H), 6.89 (d, J=8.4 Hz, 2H, Ar–H), 7.43
(s, 2H, Ar–H), 7.90 (d, J = 15.2 Hz, 2H, =CH), 7.01 (d,
J=15.6 Hz, 2H, =CH), 10.09 (s, 2H, OH), 12.188 (s, 2H,
1
1
3
COOH); C NMR: 42.88, 55.80, 78.33, 100.91, 111.94,
1
15.86,117.99, 125.57, 126.62, 147.03, 148.19, 151.30,
Table 1 The rate constant of the hydrolysis of spiroborate esters in
0% aqueous acetone at 50 °C
170.40, 176.66, 178.59.
2
In water mediated condition all spiroborate esters get
hydrolysed to yield curcumin which is clearly observable
by the decrease in absorption peak of these complexes
at its λmax and the increase in peak at 420 nm due to
the formation of curcumin. The decrease in peak at its
λmax is monitored spectrophotometrically and the rate of
decomposition observed is first order with respect to the
5
10 k (s−1)
Compound
T½ (h)
CBO
CBM
CBC
CBS
12.23
16.33
30.71
93.54
1.5
1.1
0.6
0.2
Fig. 2 Stability study of Rubrocurcumin in DMEM a after 1 h, b 2 h, c 4 h, d 24 h
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substrate. The rate constant of the hydrolysis of spirobo-
rate esters in 20% aqueous acetone at 50 °C is given in
Table 1. The Rubrocurcumin (CBO), (curcumin, boric
and oxalic acid compound) is the most stable compound
among the synthesised spiroborate esters (Fig. 2). At lower
temperature the rate of decomposition decreases and at
against the cancer cell lines used in the preliminary study
(Fig. 3d).
The complexes may have a different stability in the
medium of study and their stabilities were studied for 24 h.
The complexes were sufficiently stable for the biolological
studies condition as shown by Rubrocurcumin in the culture
medium DMEM in Fig. 2.
2
5 °C in 50% acetone the half life of CBO is 228 h and
expected to be stable in most of the biological conditions.
The Rubrocurcumin (CBO), the most stable compound
among the synthesised spiroborate esters is very active
The morphological changes observed for cancer cell
line upon addition of the synthesised spiroborate esters
(100 µg) were depicted in Fig. 3. The maximum cell death
was observed for CBO (Fig. 3d) which is found to be the
Fig. 3 Morphological changes of cancer cells upon addition of 100 µg of a CBS, b CBC, c CBM, d CBO and e cisplatin (10 µM/ml) after 24 h
of incubation
Fig. 4 Spectroflurimetric analysis of the CBO (a) and its cellular uptake (b)
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most stable in water mediated condition and selected for
further in vitro studies.
On morphological analysis, the cells did not show any mor-
phological changes (Fig. 5d).
The cytotoxicity assay results of CBO treated MCF-7
cancer cells are presented in Fig. 6. In MTT assay a con-
centration depended activity was observed. A 100 µM/ml
reduces the cell viability to 45% and at 300 µM/ml the via-
bility reduces to 32% however is less than that of standard
ant-cancer drug, cisplatin (10 mM/ml concentration of cis-
platin). Time dependent cell viability is comparatively less
indicating active drug interaction within 24 h of incubation.
Similar results are observed for NRU assay (Fig. 6b) where
Spectro flurimetric analysis of the compound
The drug CBO is fluorescent in nature as shown by the fluo-
rescence spectrum shown in Fig. 4, the maximum emission
was observed at 460 nm (Fig. 4a).
Cellular uptake studies
6
0–40% cell viability is observed for 24 h treatment of drug
Cellular uptake studies confirmed the intracellular presence
of the drug within the A431 cells (Fig. 4b), after 24 h of
incubation with cancer cell lines.
in 100 µM/ml to 300 µM/ml. The time dependence has more
influence for NRU assay than MTT assay. (Fig. 6a).
The LDH assay on MCF-7 cancer cells revealed that
there is increased release of LDH, when drug is treated with
cancer cell rather than the normal cell. For normal cell the
absorption value is < 0.1 at different times of incubation
Cytocompatibility assays in 3T3L1 adipocytes
and MCF7 and A431 cancer cells
(
Fig. 5c), however with cancer cell a concentration depended
Toxicity of CBO in normal cells 3T3L1 adipocytes are car-
ried out at different time intervals by MTT, NRU, and LDH
leakage assay and are presented in Fig. 5a–d. Studies show
that the drug is compatable with normal 3T3L1 adipocytes
as shown by more than 80% cells viability in MTT and NRU
assay and least LDH activity even after 72 h of incubation.
release of LDH is observed which ranges from 0.05 to 0.8
(Fig. 6c). The LDH release increases with the longer times
of incubation.
The morphological variations of MCF-7 cells on treat-
ment with CBO are presented in Fig. 7. The control cells
did not show any morphological changes (Fig. 7a) but
Fig. 5 Cytocompatability evaluation of the CBO on 3T3 adipocytes, a MTT assay, b neutral red assay, c LDH assay, d normal 3T3L1 adipo-
cytes, e cisplatin treated 3T3L1 adipocytes. The results of a, b and c are mean± SD, n=6, p≤0.05
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Fig. 6 Cytotoxicity assays of the CBO on MCF-7 cancer cell line. a MTT assay, b neutral red assay, c LDH assay. The results of a, b and c are
mean± SD, n=6, p≤0.05
Fig. 7 Morphological changes of MCF-7 cell line after 24 h incuba-
tion of CBO treatment: a control cells; b cells treated with curcumin
200 µM/ml CBO; e cells treated with 300 µM/ml CBO, and f cells
treated with 10 µM/ml cisplatin (positive control)
2
00 µM/ml; c cells treated with 100 µM/ml CBO; d cells treated with
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Fig. 8 Hoechst staining of MCF-7 after CBO addition a control cells; b treated with standard curcumin 200 µg/ml; c treated with 100 µg/ml
CBO; d treated with 200 µg/ml CBO; e treated with 300 µg/ml CBO; and f treated with 10 µg/ml cisplatin (positive control)
CBO treated cells were in irregular confluent aggregates
with round and polygonal cell morphology (Fig. 7c–f).
In curcumin and CBO treated MCF-7, destruction of
monolayer was observed. The treated cells with normal
polygonal morphology after 24 h of incubation, began to
shrink and became spherical in shape (Fig. 7b, e). The
cell shrinkage increased progressively in dose and time
dependent manner. This shrinkage may be due to the
growth inhibitory effect of compound.
Cell cycle analysis by FACS
When cell suspension is subjected to flow cytometry, it illu-
minates the cells when they flow individually in front of a
light source and lead to detection, counting and sorting of
the cells. In order to analyze the apoptosis induced by this
drug complex, we treated the cancer cells with CBO at dif-
ferent concentrations for 24 h.
The histogram showed that at 300 µg drug concentra-
tion, highest cell population underwent cell cycle arrest at G
0
phase (cell death occur at G phase) (Fig. 11d), confirming
0
Nuclear staining analysis—Hoechst staining
that A431cells are sensitive to the cytotoxic action of this
novel drug complex.
The images of Hoechst staining of MCF-7 cells are pre-
sented in Fig. 8 which shows that the nuclear conden-
sation pattern increases with the CBO treated cells in a
concentration depended manner. More condensation is
observed for CBO than curcumin. At 300 µg CBO addi-
tion, the cells show condensation pattern similar to the
standard drug cisplatin treated cell after 24 h (Fig. 8e).
Cytotoxicity evaluation by MTT, NRU, LDH of differ-
ent concentration of CBO addition to A431 cells shows
that at CBO-300 µg, maximum cell death was observed
Discussion
Today it is alarming to note that cancer is the record leading
cause of death across the world [3]. It is very clear that the
conventional cancer therapies result in serious side effects,
further extending patient’s life span rather giving permanent
cure. Hence, arise the demand to utilize alternative concepts
or approaches for the treatment of ‘emperor of all maladies’.
There is compelling evidence signifying the role of natu-
ral compounds in inhibiting the development and spread of
tumors in experimental animals [20]. Well standardised tra-
ditional natural products have long been used to prevent and
treat many diseases and thus they are promising candidates
for the development of anti-cancer drugs [21]. Discovery of
(
Fig. 9a–c).
The results of CBO addition at various concentrations
to A431 skin cancer cells resulted in irregular confluent
aggregates of cells with round and polygonal morphol-
ogy (Fig. 10c–e). The result was comparable to that in
cisplatin treated group (Fig. 10f).
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Fig. 9 Cytotoxicity assays of the drug on A431 skin cancer cell line. a MTT assay, b neutral red assay, c LDH assay. The results of a, b and c are
mean± SD, n=6, p≤0.05. d normal A431 cells, e Cisplatin treated A431 cells
Fig. 10 Morphological changes of A431 cell line after 24 h incuba-
tion of CBO treatment. a Control cells; b cells treated with 200 µM/
ml standard curcumin; c cells treated with 100 µM/ml CBO; d cells
treated with 200 µM/ml CBO; e cells treated with 300 µM/ml CBO;
and f cells treated with 10 µM/ml cisplatin (positive control)
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Fig. 11 Flow histogram of A431 cells added with CBO: a control; b treated with 100 µg/ml; c treated with 200 µg/ml; d treated with 300 µg/ml
(
maximum cell death at 300 µg/ml); and e quantification analysis of flow histogram
active compounds from natural products with the potential
for use as chemotherapy and/or chemotherapeutic agents are
of great interest for cancer treatment. Though chemotherapy
is largely used for treatment of cancer, it is found that the
results are not always satisfactory necessitating the need for
new and effective methods of treatment. Hence this study,
we have made an attempt to synthesise and screen novel
biostable curcumin derivative drugs (spiroborate esters of
curcumin with oxalic acid, citric acid, malonic acid and
salicyclic acid) and validate the most stable drug complex,
rubrocurmin’s (spiroborate ester of curcumin with oxalic
acid) anticancer properties in in vitro models [22].
As a hydrophobic functional food polyphenol isolated
from dried rhizomes of turmeric (Curcuma longa Linn.),
curcumin has various pharmacological activities includ-
ing anti-oxidant, anti-bacterial, anti-fungal, anti-viral,
anti-inflammatory and anti-cancer properties. Cancer is
a hyper proliferative disorder affecting the vital organs
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of the body through invasion and angiogenesis. The last
four decades have seen great medicinal advances in the
treatment of cancer exploiting different biochemical path-
ways. Cancer fundamentally alters the normal cellular
and molecular events of affected cells. These altered cells
divide and grow in the presence of signals that normally
inhibit cell growth [23] but in cancer they do not require
special signals to induce cell growth and division. Hence,
the significance of natural curcumin and curcumin con-
jugated products which could block the transformation,
proliferation, and invasion of tumor cells [24].
(10 µM/ml) based on the earlier reports proving 100% anti-
proliferative activity against cancer cell lines [28, 29].
One of the main advantages of neutral red (NR) dye is
that it readily penetrates into cell membranes through non-
ionic diffusion and the dye accumulates intracellularly in
lysosomes. Alterations of the cell surface or the sensitive
lysosomal membrane lead to lysosomal fragility and other
changes that gradually become irreversible. Such changes
result in decreased uptake and binding of NR, thereby aiding
distinction between viable, damaged, or dead cells; which is
the basis of this assay. Thus it provides a quantitative estima-
tion of the viability of cells in culture. In this study, it was
observed that NRU uptake increases with the time of incuba-
tion and the uptake was proportional to the concentration of
the NRU solution and the number of viable cells (Fig. 6b).
Lactate dehydrogenous (LDH) is a cytoplasmic enzyme
In our study, the stability of the curcumin derived drug
complexes were studied by UV–Vis spectrophotometry
and the compound, Rubrocurcumin, the spioborate ester
of curcumin with oxalic acid was found biostable in the
DMEM medium (Fig. 2). This corroborates with the earlier
reports of Bernabe-Pineda et al. [25]. In a separate report,
Souza and co-workers [26] also studied the influence of
water activity on the stability of curcuminoid pigments in
curcumin- and turmeric oleoresin–microcrystalline–cellu-
lose model systems. Wang et al. examined the degradation
kinetics of curcumin under various pH conditions and the
stability of curcumin in physiological matrices [27].
As this CBO, is a newly synthesised compound and
its cytocompatibility and anticancer effects have not been
studied earlier and since it has translational potential, we
checked initially the cytotoxicity of CBO in normal cell
line, 3LT31 adipocytes. Since we expect the efficacy of
this novel compound against the major types of skin can-
cers, we have tested the anticancer effect in two different
cell lines i.e., MCF-7 and A431, both of which have a
common epithelial origin. The anti-cancer effects of the
spiroborane complex of curcumin (Rubrocurcumin) was
confirmed from the in vitro morphology changes (Figs. 7,
+
which catalyses the inter conversion of NADH and NAD .
It converts pyruvate, the final product of glycolysis to lac-
tate when oxygen is absent or in short supply, and it per-
forms the reverse reaction during the cori cycle in the liver.
LDH released into the culture medium when cell damage or
lysis occurs during apoptosis/necrosis. LDH activity is an
indication of cell membrane integrity, otherwise an indirect
measure of cytotoxicity. LDH catalyses the reduction of a
tetrazolium salt to coloured formazan and the absorbance of
the formazan developed can be read at 490 nm. The amount
of formazan produced is proportional to the amount of LDH
released into the culture medium. In this study, we observed
that the percentage of viability in MCF-7 cells increases
with increase in concentration (Fig. 6c).
Hoechst staining is employed to study the chromatin con-
densation (Fig. 8). Nuclear chromatin condensation is one
of the hallmarks of apoptosis. Both intrinsic and extrinsic
signaling culminates ultimately in nuclear chromatin con-
densation and causes cell death. Cytotoxicity evaluation by
MTT, NRU, LDH of different concentration of CBO addi-
tion to A431 cells shows that at CBO-300 µg, maximum cell
death was observed (Fig. 10b–f).
1
0), toxicological assays such as MTT, neutral red, LDH
assays (Figs. 5, 6, 9) and nuclear chromatin condensation
by Hoechst staining (Fig. 8). On morphological analysis,
control cells did not show any morphological changes, but
CBO treated cells were observed as irregular confluent
aggregates, shrinked with round and polygonal morphol-
ogy. This shrinkage might be due to the growth inhibitory
effect of compound.
In order to analyze the apoptosis induced by the cytotoxic
agents, we treated the cells with the compound in differ-
ent concentrations for 24 h, analysed by flow cytometer and
found that cell cycle was arrested (Fig. 11). In flow cytom-
etry, the measurements were made as the cells or particles
pass through the flow cytometry, in a liquid stream. The
results showed that A431 cells are sensitive to this novel
drug complex.
MTT assay is a colorimetric method effectively used to
screen the cell proliferation and cytotoxicity of drugs. The
MTT test assesses the cell metabolism based on the abil-
ity of mitochondrial succinate hydrogenase to convert the
yellow compound, 3-(4, 5-dimethylthiazol-2-Yl)-2, 5-diphe-
nyltetrazolium bromide (MTT) to a blue formazan dye. The
amount of dye produced is proportional to the number of
live and metabolically active cells. In this study, we observed
that percentage of viability in MCF-7 cells decrease with
increase in concentration of the drug (Fig. 6a). This has
been compared with the standard cytotoxic drug, cisplatin
Another interesting observation is the fluorescent prop-
erty of this compound, which was confirmed by spectro-
flurimetric analysis. It showed the maximum emission at
460 nm (Fig. 4a). This inherent fluorescence suggests this
rubrocurmin complex as a novel candidate for targeted drug
delivery due to the easiness in in vivo tracking. In future, we
intend to incorporate this drug in nano metal like Au (gold)
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to increase its further availablity and target effectiveness.
Uptake of this fluorescent drug complex intracellularly is
a crucial data for pursuing future modifications (Fig. 4b).
Thus the results from our in vitro screening studies supports
the anti-cancer, biostable, fluorescent properties of novel
spiroborate complex of curcumin (Rubrocurcumin). Since
the drug is biostable and degrades in vivo to harmless prod-
ucts and get eliminated from body (unpublished data), we
could strongly suggest this drug as an alternate safe source
for currently used chemotherapeutics.
9. Lee WH, Loo CY, Bebawy M, Luk F, Mason RS, Rohaniza-
deh R (2013) Curcumin and its derivatives: their application in
neuropharmacology and neuroscience in the 21st century. Curr
Neuropharmacol 11:338–378
10. Wanninger S, Lorenz V, Subhan A, Edelmann FT (2015) Metal
complexes of curcumin—synthetic strategies, structures and
medicinal applications. Chem Soc Rev 7:4986–5002
1
1. Jeena J, Sudha Devi J, Balachandran SN (2017) Kinetic analysis
of thermal and hydrolytic decomposition of spiroborate ester of
curcumin with salicylic acid. Orient J Chem 33:849–858
12. Jeena J, Sudha Devi R, Balachandran S (2016) Kinetic analy-
sis of thermal decomposition of rubrocurcumin. Acta Clienta
Indica XLII C 2:121
1
3. Kornhauser A, Coelho SG, Hearing VJ (2010) Applications of
hydroxy acids: classification, mechanisms, and photoactivity.
Clin Cosmet Investig Dermatol CCID 3:135–142
Conclusion
1
4. Sui Z, Salto R, Li J, Craik C, Ortiz de Montellano PR (1993)
Inhibition of the HIV-1 and HIV-2 proteases by curcumin and
curcumin boron complexes. Bioorg Med Chem 1:415–422
The results from this in vitro study proposes the spirobo-
rate complex of curcumin, Rubrocurcumin as a novel anti-
cancer drug which is biostable and has inherent fluorescent
property. This drug with its chemotherapeutic and tracking
properties could be used as a molecular drug for site directed
therapy in cancer patients. Further pre-clinical studies in
in vivo models are warranted.
15. Asha R, Devi RS, Priya RS, Balachandran S, Mohanan PV,
Abraham A (2012) Bioactive derivatives of curcumin attenuate
cataract formation in vitro. Chem Biol Drug Des 80:887–892
1
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and survival: application to proliferation and cytotoxicity
assays. J Immunol Methods 65(1–2):55–63
17. Lasarow RM, Isseroff RR, Gomez EC (1992) Quantitative
in vitro assessment of phototoxicity by a fibroblast-neutral red
assay. J Invest Dermatol 98(5):725–9
Acknowledgements The first author acknowledges Indian Council for
Medical Research (ICMR) for the award of Senior Research fellowship
to carry out this work.
1
8. Wolterbeek HT, van der Meer AJ (2005) Optimization, applica-
tion, and interpretation of lactate dehydrogenase measurements
in microwell determination of cell number and toxicity. Assay
Drug Dev Technol 3(6):675–682
Compliance with ethical standards
19. Vijayalaksmi R, Sathyanarayana MN, Rao MVL (1981) Rubro-
curcumin reaction and its use in microdetermination of certain
organic acids. Indian J Chem 20B:907
Conflict of interest The authors confirm that there are no known con-
flicts of interest associated with this publication.
20. Amin A, Gali-Muhtasib H, Ocker M, Schneider-Stock R (2009)
Overview of major classes of plant-derived anticancer drugs.
Int J Biomed Sci 5:1–11
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1. Ji HF, Li XJ, Zhang HY (2009) Can thousands of years of
ancient medical knowledge lead us to new and powerful drug
combinations in the fight against cancer and dementia EMBO.
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