Bis(indolyl) ketohydrazide-hydrazones: Identification of
Potent and Selective Novel Tubulin Inhibitors
Reyna Valdez1, Mukund Tantak2, Dalip Kumar2 and Rachna Sadana1
1Department of Natural Sciences, UHD,
2 Department of Chemistry, Birla Institute of Technology and Science, India
Cancer is a collection of diseases hallmarked by uncontrolled cell division.
Cancer treatment involves varying combinations of surgery, radiation,
chemotherapy and hormone therapy. Chemotherapy employs the use of drugs that
kill the rapidly dividing cells (characteristic of cancer cells). Various
chemotherapeutic drugs such as paclitaxel and vinblastine interrupt cell division by
binding to tubulin (a protein responsible for spindle formation, a critical step in
cell division). For more than 50 years, tubulin binding drugs have been used to
treat cancer, Scientists are still in search of novel anti-cancer compounds targeting
tubulin for two reasons; (1) patients develop resistance to existing drugs and (2)
tubulin is one of the most validated targets for cancer treatment. Eighteen
analogues of coscinamides were screened for their cytotoxic effects on 5 different
cancer cell lines. Our initial screen identified three compounds that caused cell
death with IC50 less than 1µM. Our data indicates that the most active compounds
TMCOS-3, TMCOS-6 and TMCOS-11 induced apoptotic cell death in JURKATs
when DNA fragmentation and Caspase3/7 activation were used as apoptotic
marker assays. TMCOS-11 exerts its anti-cell proliferation activity through
inhibition of tubulin polymerization (IC50 = 20 nM) and TMCOS-11 treated cells
are stuck in G2/M phase when analyzed by flow cytometry.
Cancer is the second leading cause of deaths in the United States, and the
number of people diagnosed with cancer continues to increase every year (1).
Chemotherapy is the most commonly used regimen once cancer has spread
throughout the body. Chemotherapy drugs target rapidly dividing cells, and are
cytotoxic or cytostatic. The best results are obtained by killing cancer cells using
cytotoxic drugs rather than impeding their growth by using cytostatic drugs,
because this leave the cancer cells in a viable state. Vinca alkaloids (vinblastine)
and taxanes (paclitaxel), are commonly used anti-cancer drugs, they target tubulin,
a protein responsible for spindle organization in cell division. Although
chemotherapy has been utilized as a method for the treatment of cancer since 1940
(2), scientists are still in search of novel cytotoxic compounds because of
development of drug resistance in cancer patients, which can lead to chemotherapy
The most commonly used strategy to develop a drug involves identification
and validation of a target, selection of a lead compound form a library of
compounds by random screening, modifying the lead compound to optimize its
effects and finally, clinical trials.
Here in this study, we screened eighteen new synthetic compounds for their
cytotoxicity on five different cancer cell lines, and mechanism of cell death.
1. Mammalian Cell Culture: HCT-116, MDA-MB-231, and MCF7 cells were
maintained in DMEM complete growth media containing 10% FBS and
antibiotics (penicillin/ streptomycin), and Jurkat cells were maintained in RPMI
media with FBS and antibiotics.
2. MTT Assay: 4000-5000 cells per well seeded in a 96-well plate and MTT assay
was performed in the absence and presence of varying concentration of
compounds tested using a kit from abcam.
3. DNA Isolation: 6 X 105 cells were seeded on 35 mm dish and treated with
various compounds. Genomic DNA was isolated using a kit from abcam.
4. In Vitro Tubulin Polymeryzation: A fluorescence based assay was used to
monitor the tubulin polymerization in absence and presence of cytotoxic
compounds. A kit was purchased from Cytoskeleton Inc.
5. Caspase Assay: 100,000 cells per well were seeded in a 24-well plate in 0.5
mL media and treated with various compounds for 48 hours. 100 uL of the
sample was taken from respective wells and Caspase 3/7 assay was performed
in a 96-well plate using a commercially available kit from Promega.
6. Flow Cytometry:
Table 1. Summary of IC50 of various compounds on five different cell lines. IC50 of
TMCOS-3, TMCOS-6 and TMCOS-11 was below 0.5 uM for multiple cells lines.
• TMCOS-1, TMCOS-3, TMCOS-6, TMCOS-7 and were identified as cytotoxic
compounds in the initial screen when tested on 5 different cancer cell lines at 10
• TMCOS-3, TMCOS-6, TMCOS-7 and TMCOS-11 have IC50 less than 0.5 uM on
multiple cancer cell lines.
• TMCOS-3, TMCOS-6, TMCOS-7 and TMCOS-11 cause apoptotic cell death
when analyzed for DNA Fragmentation as apoptotic marker.
•TMCOS-3, TMCOS-6, and TMCOS-11 induced caspase 3/7 activation in
JURKAT cells, suggesting that this compounds could kill cancer cells by inducing
•TMCOS-11 inhibits microtubule formation inhibiting cell division.
•TMCOS-11 stopped ~45% of MCF7 cells at G2/M phase of the cell cycle, when
analyzed by flow cytometry.
• Natural Science Department., University of Houston-Downtown.
• ORCA grant from UHD to Dr. Rachna Sadana
1. Murphy, S., et al. (2012). Deaths: Preliminary data for 2010. National Vital
Statistics Reports, 60(4), 1-52.
2. (2000). Cancer multidrug resistance. Nature Biotechnology, 18, IT18-IT20.
BT-474 MCF-7 MDA-MB-
TMCOS-1 N.D. N.D. N.D. N.D. 3.0
TMCOS-3 N.D. 0.53 1.0 0.1 0.5
TMCOS-5 N.D. 2.3 N.D. N.D. N.D.
TMCOS-6 0.11 0.18 0.1 0.04 0.05
TMCOS-7 N.D. 1.1 N.D. 0.25 10.0
TMCOS-11 0.2 0.08 0.05 0.015 0.02
Figure 1. Illustrates the effects of 18 different compounds on cell proliferation of 5
different cancer cell lines (HCT-116, MCF7, MDA-MB-231, BT-474 and JURKAT.
Doxorubicin was used as positive control. TMCOS-3, 6 and 11 were most cytotxic.
Figure 2. JURKAT cells were treated with
various compounds as labeled on the figure
for 48 hrs. Genomic DNA was isolated and
analyzed using agarose gel electrophoresis.
TMCOS-3, 6, 7 and 11 cause cell death via
Figure 3. Dose-dependent inhibition of
tubulin polymerization by TMCOS-11.
Figure 4. Caspase activation-induced by TMCOS 3, 6, and 11 in
JURKAT cells for 48 hours. Caspase3/7 activation indicates cell
death via apoptosis.
Figure 5. Cell Cyle progression analysis in TMCOS-
11 treated MCF7 cells. a1: cell treated with vehicle,
a2: cells treated with 3 µM colchicine, a3: cells
treated with 3 µM TMCOS-11 for 24 h. b1: graph
representing % of cells in G1, S and G2/M phase. c