Cationic lipid-conjugated hydrocortisone as selective antitumor agent

Article abstract of DOI:10.1016/j.ejmech.2015.11.033

Hydrocortisone, the endogenously expressed steroidal, hormonal ligand for glucocorticoid receptor (GR), is body's natural anti-inflammatory and xenobiotic metabolizing agent. It has both palliative as well as adverse effects in different cancer patients. Herein, we show that conjugation product of C16-carbon chain-associated cationic lipid and hydrocortisone (namely, HYC16) induces selective toxicity in cancer (e.g. melanoma, breast cancer and lung adenocarcinoma) cells with least toxicity in normal cells, through induction of apoptosis and cell cycle arrest at G2/M phase. Further, significant tumor growth inhibition was observed in syngeneic melanoma tumor model with considerable induction of apoptosis in tumor-associated cells. In contrast to hydrocortisone, significantly higher anti-angiogenic behavior of HYC16 helped in effective tumor shrinkage. This is the first demonstration to convert natural hormone hydrocortisone into a selective bioactive entity possessing anti-tumor effect.

Full text of DOI:10.1016/j.ejmech.2015.11.033

Accepted Manuscript
Cationic lipid-conjugated hydrocortisone as selective antitumor agent
Bhowmira Rathore, Madhan Mohan Chandra Sekhar Jaggarapu, Anirban Ganguly,
Hari Krishna Reddy Rachamalla, Rajkumar Banerjee
PII:
S0223-5234(15)30369-X
10.1016/j.ejmech.2015.11.033
EJMECH 8223
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 April 2015
Revised Date: 31 October 2015
Accepted Date: 20 November 2015
Please cite this article as: B. Rathore, M.M. Chandra Sekhar Jaggarapu, A. Ganguly, H.K. Reddy
Rachamalla, R. Banerjee, Cationic lipid-conjugated hydrocortisone as selective antitumor agent,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.11.033.
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ACCEPTED MANUSCRIPT
Graphical Abstract

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Cationic lipid-conjugated hydrocortisone as selective antitumor agent
Bhowmira Rathorea, Madhan Mohan Chandra Sekhar Jaggarapua,b, Anirban Gangulya,b, Hari
Krishna Reddy Rachamallaa,b, and Rajkumar Banerjee*a,b
a Biomaterials Group,
CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India
* Phone: 91-40-2719-1478; Fax: 91-40-2719-3370; banerjee@iict.res.in; rkbanerjee@yahoo.com
b Academy of Scientific and Innovative Research (AcSIR), 2,Rafi Marg, New Delhi 110 001,
India.
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Abstract
Hydrocortisone, the endogenously expressed steroidal, hormonal ligand for glucocorticoid
receptor (GR), is body’s natural anti-inflammatory and xenobiotic metabolizing agent. It has
both palliative as well as adverse effects in different cancer patients. Herein, we show that
conjugation product of C16-carbon chain-associated cationic lipid and hydrocortisone (namely,
HYC16) induces selective toxicity in cancer (e.g. melanoma, breast cancer and lung
adenocarcinoma) cells with least toxicity in normal cells, through induction of apoptosis and cell
cycle arrest at G2/M phase. Further, significant tumor growth inhibition was observed in
syngeneic melanoma tumor model with considerable induction of apoptosis in tumor-associated
cells. In contrast to hydrocortisone, significantly higher anti-angiogenic behavior of HYC16
helped in effective tumor shrinkage. This is the first demonstration to convert natural hormone
hydrocortisone into a selective bioactive entity possessing anti-tumor effect.
Keywords: Glucocorticoid receptor, Hydrocortisone, cationic lipid, tumor
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Introduction:
Hydrocortisone (HYD) or cortisol is the natural, stress-induced, steroidal hormone ligand for one
of the nuclear hormone receptors namely, glucocorticoid receptor (GR). Upon HYD binding GR
classically acts as transcription factor regulating the expression of various glucocorticoid
responsive genes by acting on respective promoter elements called glucocorticoid responsive
element (GRE).1-3 This is grossly called 'GR-transactivation'. HYD as a naked drug and synthetic
glucocorticoids are used as key clinical components for the treatment of autoimmune disorders,
inflammatory diseases and specific hematologic cancers.1,4,5 Synthetic glucocorticoids are also
associated with various delivery systems to reduce inflammation around the device towards
imparting biocompatibility.6-8 However, regarding the treatment of cancer their effect is truly
limited due to the induction of refractory effects following their long-term use in cancer patients.
In contrast, HYD or its oxidized natural derivative cortisone is clinically known for long as one
of the palliative medicines for cancer patients.5 But, late evidences indicate that HYD-expression
level increases during cancer growth and may have pro-malignancy effects.9-14 GR is also linked
to various cellular proliferating factors related to cancer development.15-17 Not limited to these
but all these examples indicate that HYD and its receptor (i.e., GR) are critically involved in
various human cancer pathophysiologies. Therefore, GR and its hormone are pharmacologically
important targets for clinical interventions against cancer. However, structure-activity study
related with structural modification of HYD is critical as any irrationality in the design may
cause inadvertent, non-specific side effects.
Recently we found that as to GR-functioning, cancer cells behave differently than non-cancer
cells. Upon treating dexamethasone (i.e., Dex, a synthetic, anticancer, GR ligand)-associated
cationic liposomal gene delivery agent to different GR-expressing cells, GR-mediated
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transfection was observed selectively in only cancer cells. As a result a new form of selective
anticancer therapeutic was developed.18 Further we showed that even short chain (C8) cationic
lipid-dexamethasone conjugate could induce selective anticancer activity.19 Similar cationic lipid
conjugation of estradiol (endogenous ligand for another nuclear hormone receptor called
estrogen receptor) also yielded highly selective anti-breast cancer agent.20 All these studies
generalize that cancer cell-associated nuclear hormone receptors such as GR can be selectively
targeted by properly designed molecules, more so if it is derived from endogenous ligands such
as HYD. However, unlike Dex, unambiguous anticancer effect of naturally occurring HYD is not
established. There is also no past precedence about development of new therapeutic molecule
especially of anticancer in nature, bychemical modification of HYD as such. In the present study
we demonstrate cationic lipid modification of HYD [Scheme I] towards exhibiting highly
selective anticancer property. It was challenging because of HYD's dubious role in cancer.
Moreover, its receptor GR is also non-specifically expressed in most, if not all cells of cancer
and non-cancer lineage.21
Results and discussion
Synthesis of hydrocortisone derivatives:
As shown in Scheme I, the synthesis was easily accomplished for hydrocortisone-conjugated
cationic lipid derivatives of carbon chain lengths between 8-16 using a convergent synthetic
scheme. Firstly, the syntheses of various precursor cationic lipids were accomplished,
sequentially by incorporating twin aliphatic chains of various lengths to a mono-BOC (tert-
butyloxycarbonyl) protected ethylenediamine. This was followed by quaternization with methyl
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iodide and then converting its anion into chloride. Finally, the precursor was readied for further
reaction after BOC-deprotection (III). On the other hand, HYD was oxidized into carboxylic
acid form (IV)22, which was then reacted with cationic lipid derivatives with free amine (III) to
give respective amides and final five products of varying carbon chain lengths (V). Following
column chromatography and preparative thin-layered chromatography the desired products were
isolated, purified and characterized.
Selective toxicity of HYC16 against cancer cells
Next, the final products were then tested for their cellular toxicities against various cells. Mouse
melanoma (B16F10) cells, human cancer cells of lung (A549), breast (MCF-7), ovarian
(SKOV3), and cervical (Hela) origins were used. Side-by-side, non-cancer cells such as mouse
macrophage (RAW 264.7), mouse fibroblast (NIH3T3) and monkey kidney (COS-1) cells were
also included. Table 1 exhibits IC50s (concentration of molecules inducing 50% killing of cells)
of respective molecules in cancer and in non-cancer cells. HYC10 and HYC12 (except in A549)
induced efficient killing of both cancer and non-cancer cells, and hence were not considered as
cancer selective. The toxic effects of HYC8 and HYC14 showed confusing trend as they showed
cytotoxicity and also non-toxicity in both cancer and non-cancer cells. On the contrary, the
parent molecule HYD exhibited no cell killing effect in the given experimental condition. But
clearly, HYC16 not only showed lowest IC50s in cancer cells but also had relatively low cyto-
toxicities in non-cancer cells. This clearly exhibits HYC16's selective killing of cancer cells over
non-cancer cells. The control C16 molecule [compound IIe] showed much lower cytotoxicity in
either cancer or in non-cancer cells [Supplementary Figure S1]. Furthermore we found that the
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mixture of parent molecule HYD and C16 cationic lipid did not generate significant toxicity
towards tested cancer cells [Supplementary Figure S2]. Treatment of cationic lipid either
individually or in addition with HYD has only 20-35% toxicity at two different concentrations,
whereas, HYC16 (conjugation product of both) has > 90% toxicity in melanoma cells [Figure
S2]. This indicates that it is the chemical conjugation of the two parent molecules which is
responsible for the induced toxicity.
HYC16 selectively induces apoptosis in cancer cell
To explore the apoptosis inducing effects of the synthesized hydrocortisone-based derivatives in
normal and cancer cells, Annexin V/Propidium iodide (PI) binding based flow cytometric
apoptosis assay was performed. Incubation of A549 and B16F10 cells for 24 h with HYC16 (20
µM) significantly raised the accumulation of late apoptotic cells in cancer cells (~66% in A549,
~33% in B16F10 cells, ~40% in MDAMB-231) (Figure 1A). As expected, treatment with
equimolar concentration of hydrocortisone in the same cells had almost no response towards
inducing apoptosis. Notably, similar apoptosis inducing effects of HYC16 were almost
negligible when treated in non-cancer cells (NIH 3T3 & RAW 264.7) making it evident that
induction of apoptosis of HYC16 is justly cancer cell specific. The C16 control molecule
[compound IIe] showing no signs of apoptosis either in cancer or non-cancer cells
[Supplementary Figure S3], indicates that the apoptotic effect of HYC16 is possibly not due to
any non-specific toxic effect of C16 cationic lipid component but due to the whole molecule
HYC16.
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HYC16 exerts its toxic effect via Glucocorticoid receptor:
With a notion whether the exertion of toxicity of the synthesized hydrocortisone-based
derivatives is somehow dependent on Glucocorticoid receptor (GR) selectivity, A549 cells were
pre-treated with RU486 and then treated with HYC16. A significant enhancement of % viability
was observed compared to the cells those which were not pre-treated with RU486 (Figure 2).
This indicates that HYC16-induced cellular toxicity is GR-mediated.
HYC16 induces intrinsic pathway of apoptosis:
Since HYC16 exerted significant amount of apoptosis in murine melanoma cells, we were
interested to explore the apoptotic pathway in B16F10 cells. Compared to HYD or untreated,
HYC16 treated cell lysates showed prominent up regulation in the expression levels of some
proteins involved in intrinsic pathway of apoptosis, e.g. Bax (main pro-apoptotic protein),
cytochrome c and caspase 3 (executer protein), etc. As can be seen from (Figure 3A), the cells
upon treatment with HYC16, did not induce any significant change in the expression level of
anti-apoptotic Bcl-2 protein compared to that in cells treated with hydrocortisone (HYD).
However, prominent increase in the expression of Bax can be seen in HYC16-treated B16F10
cells compared to cells treated with unmodified hydrocortisone. The overall ratio of Bax/Bcl-2
obtained from HYC16 treated cells is almost doubled compared with that obtained from
hydrocotisone treated cells. p53 is one of the important apoptotic factors. By FACS analysis it is
revealed that p53 expression level in HYC16-treated cells was much more than in HYD or
untreated cells (Figure 3B). Clearly, HYC16 has the ability to induce intrinsic pathway of
apoptosis and also induce p53 expression to trigger apoptosis in cancer cells.
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HYC16 arrests cell cycle at G2/M phase:
To ascertain this, Propidium Iodide binding assay was performed using flow cytometry. The total
amounts of DNA present at different stages of the cell cycle, such as SubG0/G1, G1(Growth 1
phase), S(Synthesis phase) and G2/M (Growth 2 phase or the Interphase), which are denoted as
p1, p2, p3 and p4, respectively, were quantified (Figure 3C). Cells were synchronized at G1/S
boundary using double thymidine treatment, after which they were either kept untreated (UT) or
treated with HYC16 (20 µM) or hydrocortisone (20 µM).
Cells treated with HYC16 showed substantially higher DNA content in G2/M phase than in
untreated or HYD-treated cells. We finally confirmed G2/M arrest by determining the excess
accumulation of cyclin B1, which is the marker for the G2/M phase of cell cycle, with flow
cytometric analysis. Cells were synchronized with double thymidine block and treated with
HYC16 (20 µM), and HYD (20 µM) or kept untreated. Following this, the cells were subjected
to this assay. Results confirmed not only the presence of significantly enhanced populations of
G2/M phase but also higher level of cyclin B1 in HYC16-treated cells when compared with
HYD-treated or untreated cells (Figure 3D).
HYC16 effectively reduces tumor aggression:
Among all derivatives HYC16 was chosen for further tumor-biology studies. Next we developed
a melanoma subcutaneous tumor model in mice and segregated tumor-bearing mice in three
groups. Mice in different groups were respectively injected with either HYC16, HYD or kept
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untreated. As depicted in Figure 4A, HYC16 had significantly more tumor-growth inhibitory
activity than HYD at equivalent concentration. The representative tumors as isolated from each
group of sacrificed mice following the termination of experiments exhibit that the tumors from
HYC16-treated mice were smallest (Figure 4B).
To know the effect of these molecules on the health of the mice the survival data of mice over a
period of 45 days was determined. It was seen that after 45 days post inoculation of tumor cells
all untreated or 70% of the HYD treated mice were dead (Figure 4C). All HYC16 treated mice
still survived up to 45 days. Overall, this proves that the HYC16 is not only more efficient but
also the safer antitumor agent than the parent molecule, HYD.
HYC16 induces apoptosis in tumor Vasculature:
To investigate whether the drastic tumor growth reduction resulted from HYC16 induced
apoptosis of tumor vasculatures, TUNEL assay was performed on pre-fixed tumor cryosections.
To mark tumor endothelial cells, tumor-sections were stained with vascular endothelial (VE)-
cadherin-specific antibodies. Significant co-localization of the TUNEL stained areas (i.e., areas
with apoptotic bodies) and VE-cadherin (markers of tumor endothelial cells) positive cells in
tumor-sections were observed in mice treated with HYC16. This co-localization (as depicted by
yellow areas in merged images) was significantly higher with HYC16 treatment than with HYD-
treatment. Untreated mice exhibited least co-localization (Figure 5). This reveals that the tumor
growth inhibition was possibly as a result of HYC16-induced apoptosis in both tumor cells and
in tumor-associated blood vessels.
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HYC16 triggers intrinsic pathway of apoptosis in tumor lysates:
Since HYC16 showed intrinsic pathway of apoptosis in B16F10 cells in vitro we wanted to
explore ifHYC16 shows similar effect invivo. As expected the Western blot analyses of tumor
lysates showed that the apoptotic genes responsible for intrinsic pathway of apoptosis are
sequentially upregulated. Bax, cytochrome c, cleaved caspase 9, cleaved caspase 7 and also p53
expression levels were significantly increased intumors of HYC16-treated mice (Figure 6).
Bax/Bcl2 ratio was again more than twice for HYC16 treated group when compared to HYD-
treated or untreated group. This data shows that B16F10 tumor also underwent more or less
similar sequences of apoptosis as observed previously in B16F10 cells in vitro.
Anti angiogenic role of HYC16:
To show the anti-angiogenic property (if any) of HYC16 in vivo, chick embryo angiogenesis
(CEA) assay was done. In these, freshly fertilized eggs were injected with HYC16 or HYD and
the formation of blood vessels were compared with that in untreated eggs over a period of time.
The results show that HYC16ninhibits the formation of new blood vessels, whereas HYD
promotes the formation of new blood vessels as early as 4h of treatment. Interestingly, with 80
µM HYC16 treatment, not only the growths of new blood vessels are suppressed but the existing
blood vessels are also affected or damaged (Figure 7A).
Further, we investigated if HYC16-mediated tumor growth reduction was resulted due to its anti-
angiogenic role (if any). Vascular endothelial growth factor receptors (VEGFR2, a tyrosine
kinase) are avidly expressed on the surface of tumor associated vascular endothelial cells and
allow the transport of VEGF to stimulate paracrine growth within tumor microenvironment.
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Hence, any down-regulation of VEGFR2 expression signals the advent of anti-angiogenic effect.
Towards this, endothelial cells (in tumor-sections obtained from each treatment group) were
simultaneously stained with a) green fluorescent FITC-conjugated primary antibody against
VEGFR2 and b) red fluorescent VE-cadherin primary antibody (to co-stain endothelial cells).
Figure 7B shows that tumors of HYC16 treated mice exhibit significantly less VEGFR2
expression than those in the tumors of HYD-treated or untreated mice. Moreover, the density of
vascular endothelial cells observed was also comparatively less in the tumors from HYC16
treated mice than those from HYD-treated or untreated mice. These results indicate that HYC16
has clear and extensive anti-angiogenic effect and substantiate high rate of tumor-growth
inhibition in HYC16-treated tumor-bearing mice compared to HYD-treated or untreated mice.
Wound-healing assay:
To examine cell motility and proliferation of cancer cells, a scratch wound healing assay was
performed in B16F10 cells (Figure 8A). Extent of migrations of cells into the scratched are as
were analyzed. Plated cells were first scratched and then cells were either kept untreated or
treated with HYC16 or HYD. HYC16 showed much wider scratched spaces and continue to
maintain the breadth of scratch over 36h. Hydrocortisone-treated cells showed closure of scratch
by 20h whereas scratch in untreated cells was nearly closed as early as 12h from the beginning of
experiment. Quantification of relative closure of scratches (p <0.01) (Figure 8B) indicates that
HYC16 effectively barricades cancer cells from migration which reasons its effect in better
tumor regression.
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Conclusions
Collectively, we show here that the GR natural ligand HYD upon cationic lipid conjugation
exhibits potent anticancer property. Evidently, the natural ligand HYD or the cationic lipid
individually did not exhibit significant anticancer properties. Our data clearly clarifies why
cationic lipid should be conjugated to HYD rather than just being associated (as in additive
formulations). Hence, the cancer cell killing effect of the HYC16 molecule does not reflect the
additive effect of individual moieties. The anticancer property of modified HYD is selective for
cancer cells and is GR-mediated, although GR is ubiquitously expressed in all cells. Herein, we
show that natural ligand of GR, if properly modified, may still recognize cancer cell-associated
GR for and exhibit various cancer cell-selective activities. Our data clearly shows that this
selective recognition is truly useful, as the most potent molecule HYC16 can reduce tumor-
aggression. Thus, using this unique chemical molecule (HYC16) as a tool, we hereby
rediscovered that GR in cancer cells can be a potent target and hence as shown here, novel
anticancer therapeutics may be developed to treat complex diseases like cancer.
Materials and Methods:
Chemicals and General Procedures:
Hydrocortisone, EDCI, HOBT, Annexin V-FITC apoptosis detection kit and cell culture media
(DMEM) were purchased from Sigma (St. Louis, MO, U.S.). 1-bromooctane, 1-bromodecane, 1-
bromododecane, 1-bromotetradecane, 1-bromohexadecane, BOC anhydride, dioxane were
purchased from Alfa Aesar (Ward Hill, MA). All other chemicals, reagents, and organic solvents
were purchased either from Sigma (St. Louis, MO, U.S.) or from S. D. Fine Chemicals Ltd.
(Mumbai, India). The reagents were >95% pure and were used without further purification. All
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the intermediates in the synthesis were characterized by 1H NMR and/or mass spectrometry. The
1
final molecules were characterized by H NMR and ESI-HRMS, and the purity was ascertained
1
by HPLC. H NMR spectra were recorded on a Varian FT 200 MHz or Bruker FT 300 MHz
spectrometer. Chemical shifts (δ) are reported in ppm relative to chloroform resonances (1H: 7.24
ppm for [D]chloroform). Data for 1H NMR are reported as follows: chemical shift (δ in ppm),
multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quadruplet, m =
multiplet), coupling constant (Hz), integration. ESI mass spectra were obtained using a QStar XL
Hybrid QTOF mass spectrometer (Applied Biosystems). Purities of final products were clarified
with a Varian ProStar HPLC instrument at 210 nm at a flow rate of 1 mL/min in a Varian
Microsorb 100-10 BDS column (4.6 mm - 250 mm). Specific rotations were measured in Jasco
P-1020 (for HYC16) & Horiba Sepa-300 (for others) polarimetry instruments. B16F10 (murine
melanoma cells) was procured from National Center for Cell Sciences (Pune, India). Cells were
grown in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS (South American
Origin, Lonza, USA) and 1% penicillin–streptomycin– kanamycin at 37 °C in a humidified
atmosphere of 5% CO2 in air. 6–8 week old C57BL/6J mice (each weighing 20–22 g) were
purchased fromCentre for Cellular and Molecular Biology, Hyderabad, India. All the in vivo
experiments were performed in accordance with an approved animal protocol from the
Institutional Bio-Safety and Ethical Committee of CSIR-IICT. Alkaline phosphatase conjugated
goat anti-rabbit and BCIP/NBT substrate were purchased from Pierce Biotechnology (Rockford,
USA) and Calbiochem (USA) respectively. TUNEL assay kit was procured from Promega
Corporation, USA. VE-cadherin antibody and Texas red conjugated anti-mouse secondary
antibody were purchased from Santa Cruz, USA.
Chemistry.
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Synthesis of tert-butyl 2-(dihexadecylamino)ethylcarbamate [intermediate Ie (C16-analogue)]:
5g tert-butyl (2-aminoethyl) carbamate compound was dissolved in dry ethyl acetate (40 ml),
subsequently K2CO3 (17.25 g, 125 mmol) and 1-bromohexadecane (C16H33Br) (38.125 g, 125
mmol) was added. The reaction mixture was refluxed and the progress of the reaction was
monitored by TLC. After 48h reaction mixture was cooled filtered to remove K2CO3 residue.
Filtrate was diluted with ethyl acetate (200 ml), washed successively with water (3×200ml),
saturated brine (20 ml) was added and dried over anhydrous Na2SO4. Solvent was evaporated
under reduced pressure which afforded crude product which was purified by column
chromatography using 1% MeOH / CHCl3 (v/v) as the eluent to afford the tertiary amine (Ie) in
74% yield as yellow viscous oil, Rf 0.6 (5% MeOH / CHCl3).
1H NMR (300 MHz, CDCl3) : δ/ppm = 0.88 [t, 6H, CH3-(CH2)15-N-] ; 1.26-1.32 [m, 52H, CH3-
(CH2)15-N-] ; 1.42 [s, 9H,-NH-CO-O-C(CH3)3] ; 1.72 [m, 4H, {CH3-(CH2)13-CH2-CH2-}2N-CH2
–CH2-] ; 3.4 [m, 4H, {CH3-(CH2)13-CH2-CH2-}2N-CH2-CH2-], 3.6 [m, 4H, {CH3-(CH2)13-CH2-
CH2-}2N-CH2-CH2-NH-] ; 6.87 [broad s, 1H, -N-CH2-CH2-NH-].
ESI-MS: m/z = 609 (calcd. value for C39H80N2O2 = 609.0647).
Syntheses of other intermediates:
Other intermediates such as Ia-Id were similarly synthesized using the same protocol as used for
intermediate Ie except for the respective uses of 1-bromooctane, 1-bromodecane, 1-
bromododecane and 1-bromotetradecane.
Intermediate Ia: tert-butyl 2-(dioctylamino)ethylcarbamate
ESI-MS: m/z =385 (calcd. value for C23H48N2O2 = 384.63).
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Intermediate Ib: tert-butyl 2-(didecylamino)ethylcarbamate
ESI-MS: m/z = 441 (calcd. value for C27H56N2O2 = 440.74).
Intermediate Ic: tert-butyl 2-(didodecylamino)ethylcarbamate
ESI-MS: m/z = 497 (calcd. value for C31H64N2O2 =496.85).
Intermediate Id: tert-butyl 2-(ditetradecylamino)ethylcarbamate
ESI-MS: m/z =554 [M+H] (calcd. value for C35H72N2O2 =552.95).
Synthesis
of
N-(2-(tert-butoxycarbonylamino)ethyl)-N-hexadecyl-N-methylhexadecan-1-
aminium [Intermediate IIe]:
Intermediate (Ie) tert-butyl 2-(dihexadecylamino)ethylcarbamate (3 g, 4.9423 mmol) was
dissolved in methyl iodide (20 mL) and K2CO3 (2.728 g, 19.769 mmol) was added to it. The
resulting suspension was left stirred at room temperature for 12h. The reaction mixture was
filtered and the solvent was evaporated on a rotary evaporator to afford the crude residue that
was subsequently purified by column chromatography using 2% MeOH / CHCl3 (v/v) as the
eluent to yield the Intermediate IIe in 72% yield.
1H NMR (300 MHz, CDCl3) : δ/ppm = 0.88 [t, 6H, CH3-(CH2)15-N-] ; 1.26-1.32 [m, 52H, CH3-
(CH2)15-N-] ; 1.42 [s, 9H,-NH-CO-O-C(CH3)3] ; 1.72 [m, 4H, {CH3-(CH2)13-CH2-CH2-}2N-CH2
–CH2-] ; 3.35 [s, 3H, {CH3-(CH2)13-CH2-CH2-}2N+(CH3)-], 3.4 [m, 4H, {CH3-(CH2)13-CH2-
CH2-}2N-CH2-CH2-], 3.6 [m, 4H, {CH3-(CH2)13-CH2-CH2-}2N-CH2-CH2-NH-] ; 6.87 [ broad
s,1H, -N-CH2-CH2-NH-].
+
ESI-MS: m/z = 624 (calcd. value for C40H83N2O2 = 624.09).
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Syntheses of other intermediates:
Other intermediates such as IIa-IId were similarly synthesized using the same protocol as used
for intermediate IIe except for the respective uses of 1-bromooctane, 1-bromodecane, 1-
bromododecane and 1-bromotetradecane.
Intermediate IIa: N-(2-(tert-butoxycarbonylamino)ethyl)-N-methyl-N-octyloctan-1-aminium
1H NMR (300 MHz, CDCl3) : δ/ppm = 0.88 [t, 6H, CH3-(CH2)7-N-] ; 1.26-1.32 [m, 20H, CH3-
(CH2)7-N-] ; 1.42 [s, 9H,-NH-CO-O-C(CH3)3] ; 1.72 [m, 4H, {CH3-(CH2)5-CH2-CH2-}2N-CH2 –
CH2-] ; 3.35 [s, 3H, {CH3-(CH2)5-CH2-CH2-}2N+(CH3)-], 3.4 [m, 4H, {CH3-(CH2)5-CH2-CH2-
}2N-CH2-CH2-], 3.6 [m, 4H, {CH3-(CH2)5-CH2-CH2-}2N-CH2-CH2-NH-] ; 6.87 [broad s, 1H, -
N-CH2-CH2-NH-].
+
ESI-MS: m/z =399 (calcd. value for C24H51N2O2 = 399.67).
Intermediate IIb: N-(2-(tert-butoxycarbonylamino)ethyl)-N-decyl-N-methyldecan-1-aminium
1H NMR (300 MHz, CDCl3) : δ/ppm = 0.88 [t, 6H, CH3-(CH2)9-N-] ; 1.26-1.32 [m, 20H, CH3-
(CH2)9-N-] ; 1.42 [s, 9H,-NH-CO-O-C(CH3)3] ; 1.72 [m, 4H, {CH3-(CH2)7-CH2-CH2-}2N-CH2 –
CH2-] ; 3.35 [s, 3H, {CH3-(CH2)7-CH2-CH2-}2N+(CH3)-], 3.4 [m, 4H, {CH3-(CH2)7-CH2-CH2-
}2N-CH2-CH2-], 3.6 [m, 4H, {CH3-(CH2)7-CH2-CH2-}2N-CH2-CH2-NH-].
+
ESI-MS: m/z = 456 (calcd. value for C28H59N2O2 = 455.77).
Intermediate IIc: N-(2-(tert-butoxycarbonylamino)ethyl)-N-dodecyl-N-methyldodecan-1-
aminium
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1H NMR (300 MHz, CDCl3) : δ/ppm = 0.88 [t, 6H, CH3-(CH2)11-N-] ; 1.26-1.32 [m, 28H, CH3-
(CH2)11-N-] ; 1.42 [s, 9H,-NH-CO-O-C(CH3)3] ; 1.72 [m, 4H, {CH3-(CH2)9-CH2-CH2-}2N-CH2
–CH2-] ; 3.35 [s, 3H, {CH3-(CH2)9-CH2-CH2-}2N+(CH3)-], 3.4 [m, 4H, {CH3-(CH2)9-CH2-CH2-
}2N-CH2-CH2-], 3.6 [m, 4H, {CH3-(CH2)9-CH2-CH2-}2N-CH2-CH2-NH-] ;
+
ESI-MS: m/z = 512 (calcd. value for C32H67N2O2 =511.88).
Intermediate IId: N-(2-(tert-butoxycarbonylamino)ethyl)-N-methyl-N-tetradecyltetradecan-1-
aminium
1H NMR (300 MHz, CDCl3) : δ/ppm = 0.88 [t, 6H, CH3-(CH2)13-N-] ; 1.26-1.32 [m, 36H, CH3-
(CH2)13-N-] ; 1.42 [s, 9H,-NH-CO-O-C(CH3)3] ; 1.72 [m, 4H, {CH3-(CH2)11-CH2-CH2-}2N-CH2
–CH2-] ; 3.35 [s, 3H, {CH3-(CH2)11-CH2-CH2-}2N+(CH3)-], 3.4 [m, 4H, {CH3-(CH2)11-CH2-
CH2-}2N-CH2-CH2-], 3.6 [m, 4H, {CH3-(CH2)11-CH2-CH2-}2N-CH2-CH2-NH-] ; 6.2 [broad s,
1H, -N-CH2-CH2-NH-].
+
ESI-MS: m/z = 567 (calcd. value for C36H75N2O2 =567.99).
Synthesis of N-(2-aminoethyl)-N-hexadecyl-N-methylhexadecan-1-aminium [Intermediate IIIe]:
Intermediate IIe (2 g, 3.2103 mmol) was dissolved in dry DCM (10 ml) and TFA (5 ml) was
added drop wise on ice under nitrogen atmosphere and the reaction mixture was gradually
warmed to room temperature. It was stirred for 4 h to ensure complete deprotection of
tertiarybutyloxycarbonyl (BOC) group. TFA was removed completely by repeated addition of
chloroform (5 × 50 mL) followed by concentration on a rotary evaporator. Removal of the
residual solvent under high vacuum provided pure Intermediate IIIe in 70% yield as brownish
liquid. TLC Rf 0.2 (5% MeOH / CHCl3).
17

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Syntheses of other intermediates:
Other intermediates such as IIIa-IIId were similarly synthesized using the same protocol as used
for intermediate IIIe.
Synthesis
of
(8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-
carboxylic acid [Intermediate IV]:
Hydrocortisone (400 mg, 1.103 mmols) was dissolved in ethanol (78 ml) and stirred. Water (32
ml) and NaIO4 (280 mg, 1.312 mmols) were added to the solution followed by the addition of
H2SO4 (0.23 ml, 4.412 mmols) and was allowed to stir overnight at room temperature. The
reaction mixture was concentrated on a rotary evaporator. H2O (50 ml) and saturated brine (20
ml) were added to the remaining solution. 0.1 M NaOH was added until a pH of 12 was reached.
The solution was then washed 3 times with CHCl3 (100ml) and TLC was taken in 10%
methanol/chloroform. The solution was then adjusted to a pH of 3 with 1M sodium bisulfate. It
was extracted 2 times with ethyl acetate (100 ml) and a TLC was taken. It was then extracted 2-
times with (1:1) (CHCl3 : Ethyl acetate) (100ml). The organic layers were combined and dried
over sodium sulfate and solvent was evaporated under reduced pressure. Product (IV) was
obtained in 75% yield TLC Rf 0.1 (10% MeOH/CHCl3 v/v).
1H NMR (300 MHz, CDCl3): δ/ppm = 1.01[s,3H, C18H] ; 1.1-2.75 [m, 22H, for all the ring
protons and attached methyl groups with the rings(C1H+C2H+C6H+C7H+C8H+C9H+C12H
+C14H+C15H+C16H+C19H)]; 3.32 [broad s, C11H] ; 4.38 [s, C17-OH proton, 4.75 d4-MeOH [s] ;
5.65 [s, 1H vinylic proton(C4H)].
18

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ESI-MS: m/z = 349 (calcd value for C20H28O5= 348.4333).
Synthesis
of
N-(2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-
carboxamido)ethyl)-N-hexadecyl-N-methylhexadecan-1-aminium [compound V (HYC16)]:
Compound IV (2g, 5.747 mmol) was dissolved in CH2Cl2 (500 ml) followed by dimethyl
formamide (DMF) (2.3ml) to solubilize and stirred for 20 mins in ice condition.
Hydroxybenzotriazole (HOBT) (1.056 g, 6.8 mmol ) was added to this solution and stirred for ½
h. Successively 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (1.3035 g, 6.8 mmol)
was added to this solution and stirred for another ½ h. Compound (IIIe) (3.580 g, 5.747 mmol)
was dissolved in 50 ml dry CH2Cl2. Drop wise diisopropylethylamine (DIPA) was added to this
solution until the pH reached slightly basic ( at 0°C). At ice condition compound (IIIe) was
added to compound (IV). It was gradually warmed to room temperature and stirred for 15h.
Reaction mixture was washed with water (300 × 8 times ) and 2-times with 0.5N HCl followed
by saturated aqueous solution of Na2HCO3 and brine wash. It was dried over Na2SO4.
Evaporation of solvent under reduced pressure afforded the crude product which was purified by
column chromatography using 0.5% MeOH/acetone (v/v) as the eluent to afford amide (V) in
60% yield with some impurities which was finally purified with preparative TLC using 8%
MeOH/CHCl3 (2 runs). Final product with 99% purity was obtained in 15% yield, TLC Rf 0.3
(10% MeOH/acetone) and Rf 0.56 in (methanol/chloroform).
1H NMR (300 MHz, CD3OD+CDCl3): δ/ppm = 0.89 [t, 6H, CH3-(CH2)15-] ; 0.97[s,3H,C18H] ;
1.29-1.36 [m,61H, -N+ (-CH2-CH2-(CH2)13-CH3)2,C1H,C7H,C15H,C19H] ; 1.42-2.90 [m, 10H, N+
(-CH2-CH2-(CH2)13-CH3)2,C6H,C12H,C16H] ; 3.08[s,3H,CH3-N+]; 3.33 [broad s, 7H, CH3-N+ (-
19

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CH2-CH2-(CH2)13-CH3)2, C2H C11H] ; 3.56-3.66 [m, 4H, CH3-N+ -CH2-CH2-NH-]; 4.40 [s, 2H,
C11-OH,C17-OH]; 5.68 [s, 1H, C4H] ; 7.69 [s, 1H, NH].
13C NMR (300 MHz, CDCl3): δ/ppm = 9.823, 11.478, 12.209, 6.713, 3.455, 8.592, 15.597,
17.835,
25.308, 35.229, 32.977, 37.467, 41.919, 47.896, 53.889, 71.478, 107.892, 159.727, 161.824,
186.523.
+
ESI-MS: m/z = 854 (calculated mass for C55H101N2O4 = 854.4).
+
ESI-HRMS = 853.77626 (calculated Exact mass for C55H101N2O4 = 853.77559)
HPLC purity = 99.5%
[α]D25 +74 (c 1 in Dichloromethane)
Syntheses of other derivatives:
The other derivatives such as HYC8, HYC10, HYC12 and HYC14 were similarly synthesized
using the same protocol as used for HYC16 except for the respective uses of 1-bromooctane, 1-
bromodecane, 1-bromododecane and 1-bromotetradecane.
Synthesis of HYC8: N-(2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-
oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-
carboxamido)ethyl)-N-methyl-N-octyloctan-1-aminium
HYC8 was similarly synthesized using the same protocol as used for HYC16 except for the use
of intermediate (IIIa) and the final column purification was done with 3% methanol/chloroform
20

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system (v/v) as the eluent to afford amide HYC8 in 65% yield without doing further any
preparative TLC. TLC Rf 0.25 (10% MeOH/Chloroform).
1H NMR (300 MHz, CDCl3+CD3OD): δ/ppm = 0.89 [t, 6H, CH3-(CH2)7-] ; 0.96[s,3H,C18H] ;
1.26-1.31 [m,29H, -N+ (-CH2-CH2-(CH2)5-CH3)2, C1H, C7H, C15H, C19H] ; 1.47-2.27 [m, 10H,
N+ (-CH2-CH2-(CH2)5-CH3)2, C6H, C12H, C16H]; 2.41-2.84 [s, 7H, CH3-N+ (-CH2-CH2-(CH)5-
CH3)2, C2H, C11H] ; 3.10-3.14[s, 3H, CH3-N+]; 3.25-3.30 [m, 4H, CH3-N+ -CH2-CH2-NH-] ;
4.42 [s, 1H, C11-OH, C17-OH]; 5.69[s, 1H, C4H].
+
ESI-MS: m/z = 630 (calculated mass for C39H69N2O4 = 629.98).
+
ESI-HRMS = 629.4772 (calculated Exact mass forC39H69N2O4 = 629.5252).
13C NMR (300 MHz, CDCl3) : δ/ppm =14.124, 17.833, 20.883, 22.695, 26.314, 31.923, 34.890,
39.319, 49.112, 47.103, 51.398, 56.064, 62.225, 68.313, 86.172, 122.243, 172.566, 175.701,
199.661.
HPLC purity = 98%
[α]D32 + 62 (c 0.44 in DMSO).
Synthesis of HYC10: N-decyl-N-(2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-
dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthrene-17-carboxamido)ethyl)-N-methyldecan-1-aminium
HYC10 was similarly synthesized using the same protocol as used for HYC16 except for the use
of intermediate (IIIb) and the final column purification was done with 3.2%
21

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methanol/chloroform system (v/v) as the eluent to afford amide HYC10 in 63% yield without
doing further any preparative TLC. TLC Rf 0.28 (10% MeOH/Chloroform).
1H NMR (300 MHz, CDCl3+CD3OD): δ/ppm = 0.89 [t, 6H, CH3-(CH2)7-]; 0.97[s, 3H, C18H] ;
1.25-1.32 [m, 37H, -N+ (-CH2-CH2-(CH2)7-CH3)2, C1H, C7H, C15H, C19H] ; 1.43-2.08 [m, 10H,
N+ (-CH2-CH2-(CH2)7-CH3)2, C6H, C12H, C16H]; 2.41-2.83 [s, 7H, CH3-N+ (-CH2-CH2-(CH)5-
CH3)2, C2H, C11H] ; 3.09-3.15[s, 3H, CH3-N+]; 3.27 [m, 4H, CH3-N+ -CH2-CH2-NH-]; 4.44 [s,
1H, C11-OH, C17-OH]; 5.67[s, 1H, C4H].
+
ESI-MS: m/z = 686 (calculated mass for C43H77N2O4 = 686.0819).
ESI-HRMS = 685.5287 (calculated Exact mass for C43H77N2O4= 685.5878)
13C NMR (300MHz, CDCl3): δ/ppm=14.135, 17.795, 20.865, 22.366, 30.099, 31.908, 47.041,
49.142, 51.418, 56.045, 60.814, 86.279, 122.207, 199.725.
HPLC purity = 97.5%
[α]D31 +53 ( c 0.5 in Dichloromethane)
Synthesis of HYC12: N-(2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-
oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-
carboxamido)ethyl)-N-dodecyl-N-methyldodecan-1-aminium
HYC12 was similarly synthesized using the same protocol as used for HYC16 except for the use
of intermediate (IIIc)
and the final column purification was done with 3.2%
methanol/chloroform system (v/v) as the eluent to afford amide HYC12 in 67% yield without
doing further any preparative TLC. TLC Rf 0.34 (10% MeOH/Chloroform).
22

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1H NMR (300 MHz, CDCl3+CD3OD): δ/ppm = 0.89 [t, 6H, CH3-(CH2)7-]; 0.97[s, 3H, C18H] ;
1.26-1.31 [m, 45H, -N+(-CH2-CH2-(CH2)9-CH3)2, C1H, C7H, C15H, C19H]; 1.47-2.27 [m, 10H,
N+(-CH2-CH2-(CH2)9-CH3)2, C6H, C12H, C16H]; 2.6-2.7 [m, C2H, C11H]; 2.72-2.9 [t, 7H, CH3-
N+(-CH2-CH2-(CH)5-CH3)2]; 3.05[s, 3H, CH3-N+]; 3.25-3.33 [m, 4H, CH3-N+ -CH2-CH2-NH-];
4.40[s, 1H, C11-OH, C17-OH]; 5.67[s, 1H, C4H].
+
ESI-MS: m/z = 742 (calculated mass for C47H85N2O4 = 742.1883).
ESI-HRMS = 741.64771 (calculated mass for C47H85N2O4 = 741.65039)
13C NMR (300MHz, CDCl3) : δ/ppm=14.128, 17.756, 20.841, 30.929, 26.324, 29.161, 31.907,
34.826, 39.328, 47.004, 49.142, 51.462, 56.054, 60.852, 68.150, 86.337, 122.199, 172.757,
175.578, 199.711, 206.985
[α]D31 +69 ( c 0.8 in Dichloromethane).
HPLC purity = 95.1%
Synthesis HYC14: N-(2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-
2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-
carboxamido)ethyl)-N-methyl-N-tetradecyltetradecan-1-aminium
HYC14 was similarly synthesized using the same protocol as used for HYC16 except for the use
of intermediate (IIId). Column purification was done in 0.5% methanol/acetone system ( v/v) as
the eluent to afford amide HYC14 in 57% yield with some impurities which was finally purified
with preparative thin layer chromatography using 8% MeOH/CHCl3 (2 runs). Final product with
99.2% purity was obtained in 14% yield, TLC Rf 0.5 (10% MeOH/chloroform).
23

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1H NMR (300 MHz, CDCl3+CD3OD): δ/ppm = 0.88 [t, 6H, CH3-(CH2)7-]; 0.98 [s, 3H, C18H];
1.23-1.26 [m, 53H, -N+(-CH2-CH2-(CH2)11-CH3)2, C1H, C7H, C15H, C19H]; 1.43 [s, C8H, C14H];
1.41-2.02 [m, 10H, N+(-CH2-CH2-(CH2)11-CH3)2, C6H, C12H, C16H]; 3.23 [s, 3H, CH3-N+]; 3.28-
3.4 [m, 7H, CH3-N+(-CH2-CH2-(CH)5-CH3)2, C2H, C11H]; 3.67-3.88 [m, 4H, CH3-N+ -CH2-CH2-
NH-]; 4.47 [s, 1H, C11-OH, C17-OH]; 5.67 [s, 1H, C4H]; 8.62 [s, 1H, NH].
ESI-MS: m/z = 798 (calculated mass for C51H94N2O4+ = 798.29).
ESI-HRMS = 797.71403 (calculated mass for C51H93N2O4 = 797.7130)
13C NMR (300MHz, CDCl3) : δ/ppm =14.134, 17.830, 20.885, 22.701, 26.317, 29.712, 31.930,
33.402, 34.890, 39.644, 47.093, 49.121, 51.397, 56.063, 60.808, 62.227, 68.309, 86.176,
122.243, 172.589, 175.724, 199.674.
HPLC purity = 99.2%
[α]D31 +46( c 1.09 in Dichloromethane)
Biology:
Preparation of Samples and Sample Treatment.
To get a primary stock, compounds were dissolved in cell culture grade DMSO. These primary
stocks were gradually diluted with DMSO to get secondary stocks. Final working concentrations
of the derivatives were achieved by adding the secondary DMSO stocks in complete cell culture
medium containing 10% fetal bovine serum.The amount of DMSO in working solutions was
maintained not to exceed more than 1% with respect to the serum containing medium. For
cytotoxicity studies 100 ꢀL of complete media containing relevant concentrations of compounds
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was added to the each well of cells pre-seeded in 96-well plates. For estimation of apoptosis, 2
mL of complete media containing compounds in appropriate concentrations was added to the
each well of cells pre-seeded in 6-well plates.
Cell Culture:
A549 (human lung adenocarcinoma), B16F10 (mouse skin melanoma) MCF-7 (human breast
adenocarcinoma), NIH-3T3 (mouse embryonic fibroblast) and RAW 264.7 (murine macrophage)
cells were procured from National Center for Cell Sciences, Pune, India, a national repository for
cell lines/hybridomas, etc. which procures cells from American Type Culture Collection
(ATCC). All the cells were mycoplasma free. Cells were cultured in RPMI and DMEM medium
containing 10% fetal bovine serum (Lonza) and also having 50 mg/l penicillin, 50 mg/l
streptomycin and & 100 mg/l kanamycine at 37ºC in a humidified atmosphere of CO2 in air.
85–90% confluency cultures were maintained for all experiments.
Cell Cytotoxicity Studies:
To assess the Cytotoxicities of compounds 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) reduction assay was done. In a 96-well plate cells were seeded 5,000cells/well,
normally 18–24h prior to the experiment. Primary screening of toxicity of HYC8, HYC10,
HYC12, HYC14 and HYC16 (synthesized cationic derivatives of Hydrocortisone) by MTT assay
was carried out in three cancer cell lines (B16F10, A549 & MCF7) and two non-canerous cell
lines (RAW and NIH 3T3 cells) at concentrations ranging from 2.5 µM to 20 µM in DMSO. 72h
post-treatment, 10 µL of MTT solution (5 mg/mL in PBS) was added to the cells and incubated
for 4h. The formazan crystals formed were dissolved with addition of 50 µL of 1:1 (v/v)
DMSO/Methanol. Absorbances of the wells were measured with a microplate reader (ELx 800,
25

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Biotek Instruments Inc., USA) at 550 nm wavelength. Results were expressed as percent
viability = [A550 (treated cells)-background/A550 (untreated cells)-background] x 100.
Flow cytometric apoptosis analysis:
For apoptosis studies by flow cytometry, annexin V-FITC-labeled apoptosis detection kit
(Sigma) was used as per manufacturer’s instructions. Briefly, cells (2 x 105 cells/well) were
seeded in six-well plates 18-24h before treatment. Cells were either kept untreated or treated
with hydrocortisone (20ꢀM), HYC16 (20 ꢀM) for 24h.
After 24 h of treatment, cells were collected through trypsinization and resuspended in 500 µL
binding buffer containing 5 µL of annexin-V FITC and 10 µL of PI. After incubation for 15 min
in dark the samples were analysed by flow cytometer (FACS Canto II, Beckton Dickinson,
USA).
Cellular DNA Content Measurement after Cell Synchronization:
The B16F10 cells were first plated at 2x105 cells per well in T25 culture flasks. At 50-60%
confluency, the cells were washed with 1X PBS and subjected to synchronization at early S-
phase with double thymidine block and subsequent release. The cells were treated with 2 mM
thymidine diluted in DMEM containing 10% FBS for 17h, after which the cells were washed
with 1X PBS and fresh media containing 10% FBS was added and kept for 9h for the release.
The cells were again blocked for 16h with 2 mM thymidine for the second time, with a
subsequent release for 3h. After the cell synchronization, cells were kept either untreated or
treated with HYC16 (20 µm), Hydrocortisone (20 µm) or kept untreated (UT) for 18h. Cells
from different treatment groups were harvested, washed with 1X PBS and fixed with 70%
ethanol and kept at -20ºC for overnight. Cells were centrifuged to discard ethanol and re-suspend
26

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in 1X PBS. After centrifugation, cells were suspended in 1ml of PI staining solution (50 µg/ml
Propidium iodide, 0.1 mg/ml RNase, 0.05% Triton x-100) and kept in dark for 40 min at 37ºC.
After incubation, cells were collected by centrifugation, washed with 1X PBS, re-suspended in 1
ml PBS and analyzed using a flow cytometer (FACS Canto II, Becton-Dickinson, San Jose, CA,
USA) and data were analyzed with FACS Diva software. A minimum of 10000 events was gated
per sample.
Preclinical Studies:
Tumor growth inhibition study:
5-6 weeks old female C57BL/6 J mice (each weighing 20–22 g) were procured from CCMB,
Hyderabad, India. Animals were put up in an animal house maintaining institutional animal
safety guidelines. Subcutaneous injection of about ~2.5 × 105 B16F10 cells in 250 ꢀL Hank's
buffer salt solution were given in the right flank of mice on day 0. Fifteen days post-tumorcell
implantation, mice were randomly sorted out in 3 different groups (n=4) and were
intraperitonially administered with 5% glucose, HYC16 (10 mg/kg body weight) and
hydrocortisone (6.11 mg/kg body weight, equivalent to HYC16 amount). Each group received
i.p. doses on days 15, 17, 19, 21, 23, 25 post tumor inoculation. Tumor volumes (V = 1/2.ab2,
where a = maximum length of the tumor and b = minimum length of the tumor measured
perpendicular to each other) were measured with a slide calipers for up to 30-days and excised
on day 30. Results represent the means +/- SD (for n=4) (*P < 0.05 compared to control). The
experiment was aborted when average tumor volume of control treated mice was ~8000 mm3.
After termination of the experiment mice were subjected to survival study and tumor lysates
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were prepared from each treatment group. The lysates were analyzed for the expression of
apoptotic and anti-apoptotic proteins via Western blot experiment.
Histological Study:
5-6 week old female C57BL/6 J mice (each weighing 20–22 g) were procured from CCMB,
Hyderabad, India. Animals were kept in an animal house maintaining institutional animal safety
guidelines. Subcutaneous injection of about ~2.5 × 105 B16F10 cells in 250 ꢀL Hank's buffer salt
solution were given in the right flank of mice on day 0. Fifteen days post-tumor cell
implantation, mice were randomly sorted out in 3 different groups (n=4) and were
intraperitonially administered with 5% glucose, HYC16 (10 mg/kg) and hydrocortisone (6.11
mg/kg, equivalent to equimolar HYC16 amount). Each group received i.p. doses on day 15, 17,
19, 21, 23, 25 post tumor inoculation. 30th day mice were sacrificed, tumors were cryosectioned
[CM1850, Leica, Germany], fixed and immunostained for VE-cadherin (endothelial cells) and
TUNEL (apoptosis). The stained tumor slides were observed in the same positions under
fluorescent microscope (10X magnification) [Nikon, Eclipse TE2000E, Japan] using green and
red filters.
Western Blot:
B16F10 cells were seeded at a density of ~2×105 cells 18-24h pre-treatment and were treated
with HYC16 (20 µM), HYC16 (10 µM), Hydrocortisone (20 µM) or left untreated for 36h. 36h
post-treatment, the cells were detached from the flask using a cell scrapper and whole cell lysates
were prepared by treating the cells with whole cell lysis buffer (20 mM HEPES, pH 7.9, 10 mM
NaCl, 3 mM MgCl2, 0.1% Nonidet P-40, 10% glycerol, 0.2 mM EDTA, 1 mM DTT and 0.4 mM
PMSF containing 1X Protease inhibitor cocktail, Calbiochem, USA).
28