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Multitarget CDK inhibitors roscovitine and UCN‑01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription

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Colorectal cancer (CRC) is the third leading cause of cancer death in the world, and its incidence is steadily rising in developing nations. Cell cycle aberrations due to deregulation of cyclin dependent kinases (CDKs) and cyclins are common events during colorectal carcinogenesis. Herein, we investigate the anticancer potential of two multitarget CDK inhibitors viz. roscovitine (specific inhibitor of CDK1, 2, 7, and 9) and UCN-01 (pan CDK inhibitor) against three CRC cell lines. Both the drugs exerted cytotoxicity and inhibited clonogenic potential of human CRC cell lines. These drugs induced apoptosis, downregulated cell cycle regulatory and transcriptional CDKs and cyclins’ protein expression as well as their activity. Moreover, dual combination of either of these CDK inhibitors with standard chemotherapeutic drugs was found to be synergistic in CRC cells. Thus, we demonstrate that multiple CDK inhibition offers promising therapeutic strategy against CRC.
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Brazilian Journal of
Pharmaceutical Sciences
Braz. J. Pharm. Sci. 2025; 61: e24402 Page 1/23
Article
http://dx.doi.org/10.1590/s2175-97902025e24402
Multitarget CDK inhibitors roscovitine and UCN‑01
induce apoptosis in colorectal cancer cells by
inhibiting cell cycle progression and transcription
Sonal Mohan Manohar1*, Kalpana Sanjay Joshi2
1Department of Biological Sciences, Sunandan Divatia of School of Science,
SVKM’s NMIMS (Deemed-to-be) University, Vile Parle (West), Mumbai, India,
2Discovery Engine, Cipla R and D, Cipla Ltd., Vikhroli (West), Mumbai, India
Colorectal cancer (CRC) is the third leading cause of cancer death in the world, and its incidence is
steadily rising in developing nations. Cell cycle aberrations due to deregulation of cyclin dependent
kinases (CDKs) and cyclins are common events during colorectal carcinogenesis. Herein, we investigate
the anticancer potential of two multitarget CDK inhibitors viz. roscovitine (specic inhibitor of
CDK1, 2, 7, and 9) and UCN-01 (pan CDK inhibitor) against three CRC cell lines. Both the drugs
exerted cytotoxicity and inhibited clonogenic potential of human CRC cell lines. These drugs induced
apoptosis, downregulated cell cycle regulatory and transcriptional CDKs and cyclins’ protein expression
as well as their activity. Moreover, dual combination of either of these CDK inhibitors with standard
chemotherapeutic drugs was found to be synergistic in CRC cells. Thus, we demonstrate that multiple
CDK inhibition offers promising therapeutic strategy against CRC.
Keywords: CDK inhibitors. Roscovitine. UCN-01. Colorectal cancer. Cell cycle. Apoptosis. Transcription
inhibition. Drug combination.
INTRODUCTION
Colorectal cancer (CRC) is one of the most
commonly diagnosed cancer and among the leading
causes of cancer mortality in the world (Bray et al.,
2024). CRC is more frequent in developed countries.
CRC can arise spontaneously or due to family history or
due to inammatory conditions such as colitis. Extensive
metastasis is the main cause of CRC mortality within
ve years of diagnosis in about half of the patients.
Treatment is primarily surgery for localized disease,
and chemotherapy-based regimen is used for metastatic
tumors (Lee, Oh, 2016). However, drug resistance causes
relapse in most of the patients.
Deregulated cell cycle progression has been shown
to be closely associated with malignant transformation
in gastrointestinal (GI) cancers which includes CRC
(Costa, Gil Da Costa, Medeiros, 2018). In human CRC,
cell cycle abnormalities are due to hyperactivation of
CDKs and their cyclin partners along with loss of activity
of natural CDK inhibitors (Manohar, 2022). CDK1 has
been identied to be an attractive drug target for CRC
due to its constitutive activation in CRC (Salh et al.,
1999). In an integrated bioinformatics analysis study,
CDK1 along with CDC20 was shown to be strongly
correlated to poor survival in CRC patients (Li et al.,
2020). CDK1 upregulation has been observed in a subset
of human BRAF
V600E
mutant therapy- resistant colorectal
cancers (Ghafouri-Fard et al., 2022). Cyclin B1 is often
overexpressed in colorectal tumors (Korenaga et al.,
2002). Cyclin D1 is overexpressed in CRC and is also
linked to poor survival and drug/radio resistance (Maeda
et al., 1997). CDK2 overexpression is more common in
malignant CRC as compared to early lesions (Tong et
al., 2017). Cyclin A is overexpressed in about 75% of
CRCs and has been considered as a prognostic factor
for CRC (Handa et al., 1999). Cyclin B1 and cyclin E
overexpression is also linked to CRCs (Bonelli et al.,
2014).
*Correspondence: S. M. Manohar, E-mail: Sonal.Manohar@nmims.
edu; manohar.sonal@gmail.com, https://orcid.org/0000-0001-6245-9640
Braz. J. Pharm. Sci. 2025; 61: e24402Page 2/23
Sonal Mohan Manohar, Kalpana Sanjay Joshi
KRAS mutations are frequently associated with
CRC. Due to the challenges in designing direct KRAS
inhibitors, various inhibitors of upstream and downstream
pathways of KRAS have been studied in KRAS-mutant
CRCs (Ramani, Samant, Manohar, 2022; Bteich et al.,
2023). Palbociclib (CDK4/6 inhibitor), the approved
therapy for treatment of advanced breast cancers, shows
limited efcacy in KRAS-mutant CRCs when used alone
(Damato et al., 2018). It has been hypothesized that
given the hyperactivation and redundancy of many
CDKs in CRC, multitarget CDK inhibition offers more
promising treatment strategy (Manohar, 2022). Based
on the available literature, it emerges that multitarget
CDK inhibitors are more likely to hit several redundant
target CDKs simultaneously thereby reducing the risk of
unresponsiveness and drug resistance in cancer (Canavese,
Santo, Raje, 2012). Thus, in this study, we sought to
determine anticancer activity of two well-known CDK
inhibitors with differing CDK inhibitory spectrum, on
CRC cell lines viz. roscovitine and UCN-01 alone and in
combination with standard drugs in three CRC cell lines.
Roscovitine belongs to the 2, 6, 9-tri-substituted purines
and is an ATP-competitive inhibitor of CDK1, 2, 7 and
9 with almost similar IC50s against them (Meijer et al.,
1997). R-isomer of roscovitine (also known as seliciclib or
CYC202) has been shown to be more potent over racemic
roscovitine. Although results from several clinical trials
are not that promising, roscovitine is frequently used as an
experimental ‘selective’ CDK inhibitory drug in several
biological studies (Delehouzé et al., 2014). UCN-01
(7-hydroxystaurosporine) is an indolocarbazole compound
that was initially developed as protein kinase C inhibitor.
Later, it was discovered to inhibit several other kinases
including CDKs (Mull et al., 2020). UCN-01 has been
tested in several clinical trials across various types of
cancers, though its activity was not promising most likely
due to its pharmacokinetic prole (Ma et al., 2013). In the
present study, we have also scrutinized their effects on
target CDKs in order to predict the mechanism of action.
MATERIAL AND METHODS
Cell culture and reagents
Human CRC cell lines Colo-205, HCT116 and HCT-
15 were obtained from ATCC (Rockville, MD, USA).
Colo-205 and HCT-15 were cultured in RPMI-1640
medium and HCT116 in McCoy’s 5A, both the media
containing 10% fetal bovine serum (FBS) (Hyclone, UT,
USA), 2 mmol/L L-glutamine (Gibco, Grand Island,
NY, USA), 1X Antibiotic-Antimycotic solution (Gibco).
Cells were maintained at 37°C in a humidied 5% CO2
incubator. All the drugs were dissolved in dimethyl
sulfoxide (DMSO) at a concentration of 10 mmol/L (10
mM) and stored at -20°C until use; diluted in culture
medium before use and used within 4 h. All reagents
were purchased from Sigma (St. Louis, MO, USA) unless
stated otherwise.
In vitro cytotoxicity assay
Cytotoxic activity of CDK inhibitors on CRC
cell lines was assessed using CCK-8 assay (Dojindo
laboratories, Japan). Briey, cells were seeded at a density
of 3000 cells/well in 96-well plates and after overnight
incubation, treated with desired concentrations of the
drugs. At the end of the treatment, CCK-8 was added and
after 4 h incubation, absorbance was measured at 450
nm using a Spectramax Microplate Reader (Molecular
Devices, CA, USA). Data was analyzed to determine
the IC
50
(concentration of drug that inhibited cell growth
by 50%).
Clonogenic assay
Adherent CRC cell lines viz. HCT116 and HCT-15
cells were plated into six-well plates at a density of 300
cells/well and 400 cells/well respectively and incubated
overnight at 37°C in a 5% CO
2
incubator. Next day, cells
were treated with roscovitine or UCN-01 for 24 h. Medium
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Multitarget CDK inhibitors roscovitine and UCN-01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription
with drug was then replaced with medium without drug
and plates were further incubated till visible colonies
appeared. Further, cells were xed with methanol: glacial
acetic acid (2:1) and stained with 0.5% crystal violet. After
staining, plates were rinsed twice with distilled water,
dried and images were captured. Stained colonies were
counted using ImageJ software (NIH ImageJ, Biocompare,
South San Franscisco, CA, USA). Plating efciency (PE)
and surviving fraction (SF) after treatment were calculated
as per the following formulae:
PE = (no. of colonies formed/ no. of cells seeded) × 100
The number of colonies that arise after treatment of
cells expressed in terms of PE, is called the surviving
fraction.
SF= no. of colonies formed after treatment/ (no. of
cells seeded * PE)
Trypan blue viability assay
Cells were seeded at a density of 0.3 × 106 per well
into six-well plates and incubated overnight at 37°C
and 5% CO2. Next day, cells were treated with range of
concentrations of CDK inhibitors for 24 h. At the end of
the incubation period, both adherent and oating cells
were collected and washed once with PBS. Cells were
incubated at room temperature with 0.2% trypan blue in
PBS for 5 min. Samples were kept on ice, trypan blue
positive as well as total cell population were counted
microscopically using a hemocytometer and number of
viable cells was calculated.
Analysis of cell cycle distribution by ow
cytometry
CRC cell lines were seeded in T-25 tissue culture
asks at a density of 0.5 × 10
6
/mL and incubated overnight
at 37°C in a 5% CO
2
incubator. Next day, cells were treated
with or without (control) CDK inhibitors. At the end of
treatment period, cells were harvested and processed for
ow cytometry on BD FACS Aria™ (BD Biosciences,
San Jose, CA, US) (Manohar et al., 2011).
Annexin V/PI staining
Colo-205 cells were seeded at a density of 0.5 × 106
cells per T-25 ask and incubated overnight at 37°C in a
humidied 5% CO2 incubator. Next day, cells were treated
with CDK inhibitors at 2 × IC50 concentration for 24 h.
Annexin V/PI staining was performed using kit (Promega,
Madison, WI, USA) and the samples were analyzed by
ow cytometry on BD FACS Aria™ within 30 min (BD
Biosciences, San Jose, CA, US) (Manohar et al., 2019).
TdT‑mediated dUTP Nick‑End Labeling (TUNEL)
assay
HCT116 cells were seeded at a density of 0.3 ×
10
6
/well onto coverslips in six-well plate and were
allowed to adhere overnight at 37°C in a humidied
5% CO2 incubator. Next day, cells were treated with 2
× IC
50
concentrations of CDK inhibitors for 24 h and
then TUNEL assay was performed using DeadEnd
Flurorometric TUNEL system (Promega, Madison, WI,
USA) as per manufacturer’s instructions. Briey, cells
were xed using 3.7% paraformaldehyde in PBS for 25
min at 4°C and subsequently washed with PBS twice (5
min each). Then permeabilization was done using 0.2%
Triton X-100 in PBS for 5 min followed by washing with
PBS twice (5 min each). Hundred µl of equilibration
buffer was added and equilibration was carried out for
5-10 min at room temperature. Fifty µl of TdT reaction
mix was then added onto the coverslips and incubated for
1 h at 37°C in a humidied chamber in dark. Cells were
then treated with 2X SCC buffer for 15 min and washed
subsequently with PBS twice (5 min each). The coverslips
were mounted using UltraCruz mounting medium onto
glass slides and images were captured using Carl Zeiss
inverted uorescence microscope Z1.
Caspase‑Glo 3/7 assay
CRC cells were seeded (10,000 cells/well) in a 96-
well white bottom plate and were allowed to adhere
overnight at 37°C in a humidied 5% CO
2
incubator. Next
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Sonal Mohan Manohar, Kalpana Sanjay Joshi
day, cells were treated with two different concentrations
of CDK inhibitors for 24 h (roscovitine- 10, 30 µM;
UCN-01- 0.3, 1 µM). At the end of incubation, 100 µl
of Caspase-Glo 3/7 reagent (Promega, Madison, WI,
USA) was added to each well and after 1 h incubation at
room temperature; luminescence was read on PolarSTAR
Optima plate reader BMG Technologies (BMG Labtech
GmbH, Ortenberg, Germany).
Protein expression analysis by western blotting
Cells were seeded, treated with or without CDK
inhibitors, harvested at desired time points and western
blotting was carried out as previously described (Manohar
et al., 2011). Blots were developed using SuperSignal
West Femto Maximum Sensitivity Substrate (Pierce,
IL, USA) and imaged using ChemiDoc XRS+ imaging
system and image lab software (Bio-Rad Laboratories,
MA, USA). Densitometric analysis of western blots was
carried out using ImageJ software.
Antibodies used in this study were: PARP, caspase-3,
Mcl-1, Bcl-2, cyclin B1, phospho RNA Pol II CTD Ser
2/5 (Cell Signaling Technology, USA), Bcl-Xl (BD
transduction laboratories, USA), Bax, cyclin D1, CDK4,
cyclin E, cyclin A, CDK2, CDK1, CDK9, CDK7, cyclin
T1, cyclin H, phospho Rb Ser 780, total Rb, total Pol II,
anti-rabbit-IgG-HRP and anti-mouse-IgG-HRP secondary
antibodies (Santacruz Biotechnology, CA, USA), β-actin
(Sigma).
In vitro cytotoxicity assay for combination studies
CRC cells were plated in 96-well plates and treated
with standard chemotherapeutic drug or either of the
CDK inhibitors or dual combination of a standard drug
and a CDK inhibitor simultaneously for a period of 96 h.
In combination, each concentration of the standard drug
and that of CDK inhibitor were combined in a xed ratio
(as described in Results section and shown in Table SI).
Oxaliplatin concentration range selected for HCT116
was different as it was less potent as a single drug in this
cell line. Effectiveness of the combination was evaluated
on the basis of combination index (CI) calculated using
CompuSyn software wherein CI < 1, = 1, and > 1 indicate
synergism, additive effect and antagonism respectively
(Chou, 2006).
Statistical analysis
Statistical comparison was made using GraphPad
PRISM (version 8.0 GraphPad Software, Inc., USA)
software wherein Student’s paired t-test was employed.
Data are presented as mean ± SD of at least three
independent experiments. Statistical signicance was
evaluated by calculating p values and differences of p
< 0.05 were considered as statistically signicant (*p <
0.05; **p < 0.01; ***p < 0.001).
RESULT
Roscovitine and UCN‑01 exert cytotoxicity in
human CRC cells
We recently showed that the three small molecule
CDK inhibitors viz. riviciclib, roscovitine and UCN-01
exert cytotoxicity in CRC cell lines viz. Colo-205, HCT116
and HCT-15 (Manohar, Joshi, 2022). In this previous study
by us, roscovitine was found to be moderately potent
while UCN-01 was highly potent across all three CRC
cell lines with average IC50 values of 25 μM and 0.2 μM
respectively after 48 h treatment (Figure S1, Figure S2).
In the present study, we have used the same IC50 values
for assays in order to further delineate the efcacy and
mechanism of action of roscovitine and UCN-01 in CRC
cells.
Multitarget CDK inhibitors inhibit clonogenicity
and viability of CRC cells
To determine whether roscovitine and UCN-01 inhibit
the clonogenic potential, conventional clonogenic assay
was carried out using adherent CRC cell lines HCT116
and HCT-15. Plating efciency (PE) for HCT116 was 33%
with surviving fraction (SF) < 0.0001 whilst in HCT-15,
PE was 21% with SF ~ 0.002 for both drugs. Hence, both
the drugs signicantly decreased colony forming potential
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Multitarget CDK inhibitors roscovitine and UCN-01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription
of these cell lines (Figure 1B). Since this assay could not
be done in semi-adherent cell line Colo-205, cell viability
was tested in these cells after 24 h treatment with range
of concentrations of CDK inhibitors using trypan blue
dye exclusion assay (Figure 1C). UCN-01 inhibited cell
viability most potently at all concentrations (0.03-1 µM)
followed by roscovitine which elicited a dose-dependent
response at 10-100 µM.
FIGURE 1 - (A) Chemical structures of roscovitine and UCN-01, roscovitine and UCN-01 potently inhibit (B) clonogenic potential and (C)
viability of CRC cell lines.
Roscovitine and UCN‑01 abrogate cell cycle
progression in CRC cells
Cell cycle analysis of drug-treated CRC cells was
done using ow cytometry (Figure 2). At 6 h in Colo-
205, UCN-01 decreased number of cells in S phase in
comparison to untreated control and increased in sub
G1 which signies cell death. No signicant changes in
the cell cycle distribution were seen with roscovitine in
this cell line at 6 h. At the same timepoint, In HCT116,
roscovitine decreased G0-G1 fraction and UCN-01 lead
to reduced G0-G1 fraction and signicantly increased
sub G1 fraction. At 6 h, in HCT-15, roscovitine 3 × IC50
increased sub G1 fraction and UCN-01 showed decreased
G0-G1 with increase in sub G1 at 6 h for both IC50 and 3
× IC50. After 18 h, increase in cell death with roscovitine
and UCN-01 was signicant. At a later time point of 24
h, HCT-15 was least sensitive to both the drugs and the
pattern of cell cycle distribution was similar to that seen
at 18 h treatment.
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Sonal Mohan Manohar, Kalpana Sanjay Joshi
FIGURE 2 - Effect of CDK inhibitors roscovitine and UCN-01 on the cell cycle in CRC cells. (A) Representative data of cell cycle analysis after
24 h treatment. (B) Percent population of CRC cells in sub G1 (indicating apoptosis) after drug treatment.*: p < 0.05; **: p < 0.01; ***: p < 0.001.
CDK inhibitors induce apoptosis in CRC cells
Apoptosis induction by CDK inhibitors in CRC cell
lines was conrmed using annexin V/PI, TUNEL and
Caspase-glo 3/7 assays. Twenty-four hours timepoint was
selected which is equivalent to one complete cell cycle and
concentration of 2 × IC
50
was used. Signicant apoptosis
was induced in Colo-205 as evident from signicantly
increased annexin V positive-PI positive population after
roscovitine or UCN-01 treatment (p < 0.01) (Figure 3A).
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Multitarget CDK inhibitors roscovitine and UCN-01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription
FIGURE 3 - Apoptosis-inducing potentital of roscovitine and UCN-01 in CRC cell lines is conrmed by: (A) annexin V/PI staining in Colo-205
(B) TUNEL assay in HCT116 and (C) caspase-3/7 enzyme activity assay in HCT116 and HCT-15.
Terminal deoxynucleotidyl transferase–mediated nick
end labeling (TUNEL) assay was employed to conrm
drug-induced apoptosis in HCT116. When compared
with the control, treated cells were positively labeled by
TUNEL and nuclear fragmentation in these apoptotic
cells was also detectable when counterstained with DAPI
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Sonal Mohan Manohar, Kalpana Sanjay Joshi
(Figure 3B). Thus, it was evident that roscovitine and
UCN-01 induced apoptosis in this cell line.
Further, Caspase-glo 3/7 assay was used to conrm
increase in caspase-3/7 activity upon CDK inhibitor
treatment- a hallmark of apoptosis. Both the CDK
inhibitors signicantly increased caspase-3/7 activity
in both cell lines. UCN-01 showed remarkable caspase
activation in HCT116 (upto 6 fold) (Figure 3C). Overall,
increase in caspase-3/7 activity was signicantly more
in HCT116 as compared to HCT-15.
After conrming apoptosis by the above mentioned
cell-based assays, effect of roscovitine and UCN-01 was
tested on apoptotic markers i.e. cleaved caspase-3, cleaved
PARP (substrate for caspase-3) and Bcl-2 family proteins
after 24 h drug treatment. There was a signicant increase
in cleaved capsase-3 levels in response to treatment with
both drugs in all the three cell lines (Figure 4). Cleaved
PARP levels were found to be signicantly increased by
both the drugs in HCT116 and HCT-15. In Colo-205,
there was no detectable increase in cleaved PARP upon
roscovitine treatment. Mcl-1 was downregulated most
signicantly in HCT116 by both drugs while Bcl-2 was
downregulated most potently in HCT-15 by both drugs.
Bcl-Xl levels remained unchanged in all three cell lines
upon drug treatment. Bax levels were unchanged in Colo-
205, slightly increased in HCT116 and slightly decreased
upon treatment in HCT-15. Densitometric analysis of all
western blots is given in Figure S3.
FIGURE 4 - Roscovitine and UCN-01 induce PARP and caspase-3 cleavage and downregulate anti-apoptotic proteins in CRC cell lines. Control:
untreated, Rosco: roscovitine, x: IC50, 3x: 3 × IC50, Cl: cleaved, Casp-3: caspase-3.
Roscovitine and UCN‑01 modulate expression of
cell cycle regulatory proteins
Changes in protein levels of cell cycle regulatory
CDKs and cyclins in response to treatment with CDK
inhibitors were analyzed by western blotting in HCT116
(Figure 5A-C). Both the drugs downregulated cyclin D1
and CDK4 protein levels. Surprisingly, cyclin E was up
regulated by roscovitine at IC
50
concentration at 18 h and
24 h time points. As anticipated, cyclin A expression was
inhibited by roscovitine whereas CDK2 levels remained
almost unchanged. Whilst cyclin B1 was downregulated
after 18 h and 24 h, CDK1 remained almost unchanged
in response to roscovitine treatment.
Cyclin E levels were downregulated by UCN-01
after 24 h treatment with both concentrations. UCN-01
also increased cyclin A levels after 6 h with 3 × IC
50
followed by signicant downregulation after 18 and 24
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Multitarget CDK inhibitors roscovitine and UCN-01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription
h treatment with both concentrations. This drug led to
down regulation of CDK2, CDK1 and cyclin B1 at all
three time points at both concentrations.
Overall, results indicate that CDK inhibitors down
regulated cell cycle-related cyclins and CDKs with varied
potency in HCT116 cell line.
FIGURE 5 - Effect of (A) roscovitine and (B) UCN-01 on expression of cell cycle-related CDKs and cyclins in HCT116. Well no. 1: untreated,
2: treated with IC50, 3: treated with 3 × IC50.
Roscovitine and UCN‑01 inhibit transcription
Previously, it has been reported by us and others
that CDK inhibitory drugs inhibit transcriptional CDKs
and cyclins in hematological cancers (MacCallum et
al., 2005; Manohar et al., 2011). In the present study,
both the drugs downregulated cyclin T1, CDK9, and
CDK7 protein levels after 18 h treatment in HCT116
cells. Unpredictably, cyclin H levels were found to be
upregulated by roscovitine IC
50
and UCN-01 3 × IC
50
(Figure 6A).
Both roscovitine and UCN-01 were shown to potently
inhibit phosphorylation of Rb at Ser 780 (cell cycle CDK
substrate) and RNA polymerase II carboxy-terminal
domain at Ser 2/5 (RNA Pol II CTD) (transcriptional CDK
substrate) after 18 h treatment in HCT116 (Figure 6B).
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Sonal Mohan Manohar, Kalpana Sanjay Joshi
FIGURE 6 - Roscovitine and UCN-01 downregulate protein expression of (A) transcriptional CDKs and cyclins and (B) phospho-Rb and
phospho-RNA Pol II CTD in HCT116. Control: untreated, Rosco: roscovitine, x: IC50, 3x: 3 × IC50.
CDK inhibitors are synergistic with standard
chemotherapeutic drugs against CRC cells
Currently, standard-of-care for CRC includes
chemotherapeutic drugs viz. 5-FU, irinotecan and
oxaliplatin. Efficacy of dual combination of these
standard drugs with CDK inhibitors in CRC cell lines
was tested using CCK-8 cytotoxicity assay. Each of the
standard chemotherapeutic drug was combined with a
CDK inhibitor in a xed ratio which was based on the
IC50 values of the single drugs (Chou, 2006) (Table SI).
Average 72 h IC50 values of standard chemotherapeutic
drugs were: 5-FU: 6 μM, oxaliplatin: 0.8 μM (for HCT-
15) and 5.5 μM (for HCT116). The drugs were added
together for 96 h. Effectiveness of the combination was
evaluated using combination index (CI) calculated by
the CompuSyn software (CI < 1, = 1, and > 1 indicate
synergism, additive effect and antagonism respectively).
Remarkably, both CDK inhibitors showed synergism
with at least one of the standard drugs in CRC cell lines
as described below.
Colo‑205
UCN-01 was synergistic when added together with
5-FU (CI = 0.15-0.94) or with irinotecan (CI = 0.21-
0.71). Roscovitine showed synergism when added with
oxaliplatin (CI = 0.09-0.8) (Figure 7A).
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Multitarget CDK inhibitors roscovitine and UCN-01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription
FIGURE 7 - Combination indices (CI) of synergistic dual combinations of roscovitine or UCN-01 with standard drugs in CRC cell lines. Values
are presented as mean ± SD of three independent experiments, each in triplicates. Irino: irinotecan, Oxalo: oxaliplatin, 5-FU: 5-uorouracil.
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Sonal Mohan Manohar, Kalpana Sanjay Joshi
HCT116
Notably, UCN-01 was very strongly synergistic in
combination with oxaliplatin (CI= 0.03–0.77) and so
was roscovitine (CI= 0.2-0.9) (Figure 7B). UCN-01 was
moderately synergistic with 5-FU (CI= 0.4–0.7).
HCT‑15
In this multidrug resistant cell line, UCN-01 was
moderately synergistic with oxaliplatin (CI= 0.16–1.18)
(Figure 7C).
DISCUSSION
Aberrant activation of cell cycle-related oncogenic
proteins in human colorectal cancer offers prospects
for anticancer drug discovery. In the present study,
we compared the efcacy of two CDK inhibitors with
differential CDK inhibitory spectrum in three CRC cell
lines. Both the drugs exerted potent anticancer activity
irrespective of their specicity for target CDKs. UCN-01
being a non-specic (pan CDK) inhibitor was more potent
with almost 10 times lower IC50 than that of roscovitine
which is specic for CDK1, 2, 7 and 9. Both drugs
effectively restrained clonogenic capacity of CRC cells
which indicates loss of tumor cell reproductive viability.
We also conrmed the apoptosis-inducing potential of
these inhibitors using cell cycle analysis, annexin V/PI
assay, TUNEL assay and Caspase-glo 3/7 assay in CRC
cell lines.
Most of the rst generation CDK inhibitors were
less selective, these drugs affected several CDK family
members (Blagosklonny, 2004). Although these broad
spectrum CDK inhibitors showed good cytotoxicity in vitro,
many of them failed in clinical trials as single agents due to
toxic effects because of their lack of specicity. Targeting
cell cycle regulatory CDKs along with transcriptional
CDKs has been shown to induce apoptosis in various
types of cancer cells (Shirsath, Manohar, Joshi, 2012).
Yet, the spectrum of CDKs to be targeted for optimum
efcacy would highly vary for every type of cancer (Graf,
Wuest, Pietzsch, 2011; Joshi et al., 2012). Based on
the literature, it is hypothesized that a multitarget CDK
inhibitor increases the likelihood of hitting a particular
target CDK in the case of a heterogeneous disease like
cancer. Several of the CDK inhibitors currently under
clinical development are essentially CDK1, CDK2, CDK7
and CDK9 inhibitors, in addition to CDK4/6 inhibitors
in the pipeline. Other kinases outside the CDK family
are also inhibited by some of these drugs (Galons et al.,
2013). It has become increasingly apparent that such
agents act through mechanisms other than or in addition
to cell cycle arrest viz. apoptosis and anti-angiogenesis
(Krystof, Baumli, Fürst, 2012; Manohar, Joshi, 2022;
Manohar, Joshi, 2023).
In the present study, roscovitine and UCN-01
abrogated expression of not only cell cycle regulatory
cyclins and CDKs but also transcriptional cyclins and
CDKs along with their substrates viz. phospho Rb
and phospho-RNA Pol II CTD. Therefore, efcacy of
roscovitine and UCN-01 against CRC cells can be clearly
attributed to their CDK1, CDK2, CDK4, CDK7 and
CDK9 inhibitory activity although they were found to
only moderately affect CDK2 and cyclin E and cyclin
A expression in the present study. These results are in
line with previous work by others (Senderowicz, 2002;
Whittaker et al., 2004; Crescenzi, Palumbo, Brady, 2005).
Roscovitine is an orally bioavailable small molecule
inhibitor of several CDKs competing at their ATP-
binding sites. Previous studies indicate that, it inhibits
Rb phosphorylation and it also decreases expression of
cyclin D1, A and B1 probably through loss of RNA Pol
II phosphorylation (Whittaker et al., 2004). Roscovitine
has been shown to have signicant antitumor activity in
HCT116 and LoVo human colon cancer xenografts models
(79% and 45% tumor growth inhibition respectively)
(Raynaud et al., 2005). Synergistic effect of roscovitine
with conventional cytostatic drugs such as gemcitabine,
doxorubicin and cisplatin has been reported (Crescenzi,
Palumbo, Brady, 2005; MacCallum et al., 2005; Raje
et al., 2005). It has been shown to induce apoptosis via
short-lived anti-apoptotic protein Mcl-1 down regulation
through RNA Pol II-dependent transcription inhibition in
multiple myeloma cells and mantle cell lymphoma cells
Braz. J. Pharm. Sci. 2025; 61: e24402 Page 13/23
Multitarget CDK inhibitors roscovitine and UCN-01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription
(MacCallum et al., 2005). In fact, transcription inhibition
and induction of apoptosis via Mcl-1 downregulation
has been suggested to be the key mechanism that is
attributable to the anticancer activity of roscovitine in
these cell types. In the present study, roscovitine-induced
apoptosis of CRC cells can be certainly attributed to Mcl-1
downregulation since Mcl-1 plays a key role in CRC cell
survival and is known to mediate drug resistance in CRC
cells (Manohar, Joshi 2022). In fact, it has been proposed
that inhibition of RNA Pol II activity by roscovitine leads
to downregulation of a large no. of genes which leads
to rapid onset of apoptosis in cancer cells (Delehouzé et
al., 2014).
UCN-01 is a staurosporine derivative isolated from
Streptomyces (Senderowicz, 2002). UCN-01 induces cell
cycle arrest in a specic phase of cell cycle depending on
the cell type. It was shown to lead to S and G2/M arrest
in hepatocellular carcinoma cells (Wu et al., 2013) and
was also shown to induce DNA damage and autophagy
in osteosarcoma cells (Lien et al., 2018). Roscovitine has
also been shown to induce cell cycle arrest at S phase and
G2/M transition and apoptosis at higher concentrations
(Crescenzi, Palumbo, Brady, 2005). In the present study,
both roscovitine and UCN-01 directly led to increase in sub
G1 population in all three CRC cell lines. Among the CRC
cell lines, HCT-15 was found be slightly less sensitive
to both the drugs (w.r.t. increase in sub G1 fraction in
treated cells), this could be attributed to inherent multidrug
resistant feature of this cell line. Literature suggests that
transcription inhibitors lead to p53-independent apoptosis
in cancer cells, hence it is unlikely that HCT-15 is less
sensitive to CDK inhibitors used in this study owing to
its p53 mutant status (Gartel, 2008). Previously, it has
been shown that UCN-01 downregulates Bcl-Xl in CRC
cell lines (LS513 and SW48) thereby inducing apoptosis
(Bhonde et al., 2005). However, in the present study,
we observed that Bcl-Xl levels did not change after
treatment with UCN-01 in Colo-205 and HCT-15 and
only Bcl-2 downregulation was observed. Moreover,
Mcl-1 expression was inhibited to much lesser extent in
HCT-15 as compared to Colo-205 by roscovitine and not
altered at all in HCT-15 by UCN-01. This could be due
to much higher basal expression of Mcl-1 in HCT-15.
We recently showed that in addition to the action
against cell cycle regulatory proteins and apoptotic
proteins, multitarget CDK inhibitors also exhibit off-target
activity against other aberrant signaling pathways in CRC
cells such as Ras/MAPK and PI3K/Akt. Further, these
drugs exhibit potent anti-migratory and anti-angiogenic
potential which certainly contributes to their potency in
CRC cells (Manohar, Joshi 2023).
Combination therapy has been the standard-of-care
in cancer treatment that aims to increase fractional cell
kill using suboptimal concentrations of two or more drugs
leading to improved overall response. In the in vitro
studies, concentration and duration of administered drugs
can be tightly controlled and the inhibition of tumor cell
growth can be easily measured (Chou, 2006). Inclusion
of CDK inhibitors in combination therapeutic regimens
has been proposed to be a potential strategy to increase
their potency against cancers including CRC (Chou 2006;
Rathos et al., 2012; Rathos et al., 2013). Recently, it was
shown that multiple CDK inhibition enhances cytotoxicity
of 5-FU in colon cancer cells by upregulation of DR5
via p73 (Tong et al., 2023). In the present study, both
roscovitine and UCN-01 were found to be synergistic
with at least one of the standard drugs in CRC cell lines.
Yet, these drugs exhibited limited synergism in HCT-15
perhaps owing to multidrug resistant nature of this cell
line.
Overall, our ndings implicate that multitarget CDK
inhibitors roscovitine and UCN-01 potently abrogate
cell cycle progression in CRC cells and also inhibit
transcription thereby inducing signicant apoptosis.
Further, both the drugs are synergistic with standard drugs
used for CRC treatment in in vitro combination studies.
This study suggests that multitarget CDK inhibitors may
serve as effective anticancer agents against CRC even in
the therapy resistant setting.
Braz. J. Pharm. Sci. 2025; 61: e24402Page 14/23
Sonal Mohan Manohar, Kalpana Sanjay Joshi
ACKNOWLEDGMENTS
We would like to thank Piramal Life Sciences,
Goregaon, Mumbai, India for supporting this research
work and for providing necessary permissions to publish
the same. We are grateful to the anonymous reviewers
for improving quality of this article by providing their
valuable inputs.
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Received for publication on 21st May 2024
Accepted for publication on 18th July 2024
Associated Editor: Silvya Stuchi Maria-Engler
Braz. J. Pharm. Sci. 2025; 61: e24402 Page 17/23
Multitarget CDK inhibitors roscovitine and UCN-01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription
SUPPLEMENTARY INFORMATION
FIGURE S1 - Comparative 48 h and 72 h IC50 of roscovitine and UCN-01 in CRC cell lines.
Colo‑205
FIGURE S2 - Roscovitine and UCN-01 inhibit survival of CRC cell lines.
Braz. J. Pharm. Sci. 2025; 61: e24402Page 18/23
Sonal Mohan Manohar, Kalpana Sanjay Joshi
HCT116
HCT‑15
For blots in Figure 4
Braz. J. Pharm. Sci. 2025; 61: e24402 Page 19/23
Multitarget CDK inhibitors roscovitine and UCN-01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription
Braz. J. Pharm. Sci. 2025; 61: e24402Page 20/23
Sonal Mohan Manohar, Kalpana Sanjay Joshi
Braz. J. Pharm. Sci. 2025; 61: e24402 Page 21/23
Multitarget CDK inhibitors roscovitine and UCN-01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription
For blots in Figure 5
Braz. J. Pharm. Sci. 2025; 61: e24402Page 22/23
Sonal Mohan Manohar, Kalpana Sanjay Joshi
For blots in Figure 6
FIGURE S3 - Densitometric analysis of western blots. N=3, bars indicate mean ± SD of three independent experiments.
Braz. J. Pharm. Sci. 2025; 61: e24402 Page 23/23
Multitarget CDK inhibitors roscovitine and UCN-01 induce apoptosis in colorectal cancer cells by inhibiting cell cycle progression and transcription
TABLE SI - Ratios of drug combinations
Roscovitine UCN-01
5-uorouracil 1:1 10:1
Irinotecan 1:3 1:0.3
Oxaliplatin (Colo-205,HCT-15) 1:3 1:0.3
Oxaliplatin (HCT116) 1:1 1:0.3
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Colorectal cancer (CRC) is a common cancer in the world and its prevalence is increasing in developing countries. Deregulated cell cycle traverse is a hallmark of malignant transformation and is often observed in CRC as a result of imprecise activity of cell cycle regulatory components, viz. cyclins and cyclin-dependent kinases (CDKs). Apart from cell cycle regulation, some CDKs also regulate processes such as transcription and have also been shown to be involved in colorectal carcinogenesis. This article aims to review cyclin-dependent kinases as potential targets for CRC. Furthermore, therapeutic candidates to target CDKs are also discussed.
Article
Background Colorectal cancer (CRC) is the third leading cause of cancer death worldwide, and its incidence is steadily rising in developing nations. Cell cycle aberrations due to deregulation of cyclin dependent kinases (CDKs) and cyclins are common events during colorectal carcinogenesis. Yet, efficacy of multitarget CDK inhibitors as therapeutic agents has not been much explored against CRC. Objective The anticancer potential of multitarget CDK inhibitor riviciclib (also known as P276-00), was investigated against CRC cell lines of varied genetic background. Method Cytotoxicity of riviciclib - potent CDK1, CDK4 and CDK9-specific inhibitor was evaluated in vitro. Further, its effect on clonogenic potential, cell cycle, apoptosis and transcription was tested using colony forming assay, flow cytometry and western blot analysis respectively. Also, efficacy of riviciclib in combination with standard chemotherapeutic agents was assessed. Dependency of CRC cells on specific CDKs for their survival was confirmed using siRNA studies. Results Riviciclib exerted significant cytotoxicity against CRC cells and inhibited their colony forming potential. It induced apoptosis along with inhibition of cell cycle CDKs and cyclins as well as transcriptional CDKs and cyclins. Moreover, dual combination of riviciclib with standard chemotherapeutic drugs exhibited synergism in CRC cells. siRNA studies indicated that CRC cells are dependent on specific CDKs for their survival which are targets of riviciclib. Conclusion This study provides evidence that multitarget CDK inhibitors can serve as promising therapeutic agents against CRC alone or in combination.