Evaluating the ability of anti-cancer drugs Etoposide and Staurosporine to induce apoptosis in HeLa cells as a potential polytherapy for cervical cancer patients
Cervical cancer is considered one of the most prevalent cancers affecting Singaporean women.
Although many novel chemotherapeutics have been developed recently, little has been done to
determine the efficiency of current anti-cancer agents working in combination. Here, we aimed to
evaluate the apoptosis induction efficiency of Etoposide and Staurosporine in HeLa cells. Cell cultures
were subjected to either 50 μM Etoposide, 10 nM Staurosporine or both for 24 hours prior visualization
under a fluorescence microscope. We found that Etoposide alone had an efficiency of 16.1% while
Staurosporine alone had 18.3%. However, the polytherapy achieved an efficiency of up to 33.6%,
which indicates an additive effect of both drugs to induce apoptosis. Our results demonstrate that
Etoposide and Staurosporine are both capable of inducing apoptosis in HeLa cells. Furthermore, it
reveals the potential of Etoposide-Staurosporine polytherapy to be a potent combinative treatment
option for cervical cancer patients resistant or sensitive to conventional anti-cancer agents.
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Evaluating the ability of anti-cancer drugs Etoposide and Staurosporine to induce apoptosis in HeLa cells as a potential polytherapy for cervical cancer patients
1. NANYANG TECHNOLOGICAL UNIVERSITY
BS3004 – CANCER BIOLOGY
Evaluating the ability of anti-cancer drugs Etoposide and
Staurosporine to induce apoptosis in HeLa cells as a potential
polytherapy for cervical cancer patients
Member Name E-mail
1 Balakumaran S/O
Nadarajan
B150018@e.ntu.edu.sg
2 Goh Jun Wei JGOH052@e.ntu.edu.sg
3 Jessica Ng Pei Zhen JNG090@e.ntu.edu.sg
4 Srija K Nair SRIJA001@e.ntu.edu.sg
Date Submitted: 28 September 2016
2. Abstract
Cervical cancer is considered one of the most prevalent cancers affecting Singaporean women.
Although many novel chemotherapeutics have been developed recently, little has been done to
determine the efficiency of current anti-cancer agents working in combination. Here, we aimed to
evaluate the apoptosis induction efficiency of Etoposide and Staurosporine in HeLa cells. Cell cultures
were subjected to either 50 µM Etoposide, 10 nM Staurosporine or both for 24 hours prior visualization
under a fluorescence microscope. We found that Etoposide alone had an efficiency of 16.1% while
Staurosporine alone had 18.3%. However, the polytherapy achieved an efficiency of up to 33.6%,
which indicates an additive effect of both drugs to induce apoptosis. Our results demonstrate that
Etoposide and Staurosporine are both capable of inducing apoptosis in HeLa cells. Furthermore, it
reveals the potential of Etoposide-Staurosporine polytherapy to be a potent combinative treatment
option for cervical cancer patients resistant or sensitive to conventional anti-cancer agents.
1. Introduction
1.1 Human Cervical Carcinoma
Cervical cancer is one of the top ten most prevalent cancers affecting Singapore’s female
population [1]. Over fifty decades, the HeLa cancer cell line has been used to study the disease
mechanism and evaluate potential anti-cancer therapeutics in vitro.
HeLa cells were first isolated from a patient, Henrietta Lacks, who was diagnosed with the
cancer in 1951 [2][3]. The etiology was unclear until further studies revealed that high-risk human
papillomaviruses (HPV), such as oncogenic type 16 and 18, were potent cervical carcinoma induction
agents [4]. Viral oncogene product E6 binds and targets p53 protein for proteasomal degradation while
E7 binds and inhibits hypophosphorylated retinoblastoma protein (pRb), releasing pRb-bound E2F-1
transcription factor [4]. Furthermore, E5, E6 and E7 gene products have been shown to attenuate the
phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling cascade,
promoting tumor initiation and progression [5]. Sustained expression of these viral oncogenes confer
infected cells the ability to bypass cell cycle checkpoints and divide indefinitely [4][5].
1.2 Etoposide
DNA Topoisomerase II α (TopoIIα) induces a double-strand break (DSB) in the DNA to
unwind the duplex for processes such as mRNA transcription or DNA replication [6][7]. Although the
TopoIIα DSBs are transient and DNA strands are re-ligated, Etoposide bound to TopoIIα cleavage
complex inhibits this re-ligation [7]. Consequently, multiple single- and double-strand DNA breaks
could trigger cell cycle arrest or the p53-dependent intrinsic apoptosis pathway [6][7].
As TopoIIα is overexpressed in tumour cells but not in normal quiescent cells, Etoposide is
being used as a tumour specific chemotherapeutic agent [6]. However, Etoposide treated HeLa cells
ability to undergo apoptosis despite the lack of functional p53 proteins reveal that TopoIIα may not be
the only drug target and p53-independent apoptotic pathways may be initiated [7].
3. 1.3 Staurosporine
Staurosporine is a broad-spectrum protein kinase inhibitor that triggers apoptosis slightly
different from DNA-damaging anti-cancer drugs like Etoposide [8]. Some prominent inhibitory targets
are Akt, protein kinase C (PKC) and I-kappa B kinase (IKB), which inhibition decreases anti-apoptotic
factors activation and/or synthesis, while keeping pro-apoptotic factors such as Bcl-2-associated death
promoter (Bad) and caspase-9 active [8][9].
In addition to the conventional intrinsic apoptosis pathway involving cytochrome c and
apoptotic proteases activating factor 1 (Apaf-1) that mediates caspase-9 activation, Staurosporine is
able to activate caspase-9 independently through a novel pathway [9]. Although a promising anti-
cancer drug, its toxic effects limits clinical applications [10]. However, as Staurosporine mechanism of
action differs partially from Etoposide, a co-administration with other chemotherapeutic agents may
yield increased efficacy with reduced side effects.
1.4 Fluorescence-based Apoptosis Assay
Differentiating apoptotic cells from normal and necrotic cells is challenging. However, a unique
characteristic of cells undergoing apoptosis is the activation of executioner caspases-3, -6 and -7 as an
endpoint event [11]. The IncuCyteTM
Caspase 3/7 Reagent (CasR) is a kinetic fluorescence assay that
leverages on this unique property. As CasR possess a bound DNA intercalating dye via a DEVD linker
cleavable by caspases-3 and -7, the dye is able to fluorescently label only nuclear DNA of apoptotic
cells [12]. Together with CasR, we aimed to evaluate the ability of Etoposide and Staurosporine to
induce apoptosis in HeLa cells.
2. Materials and Methods
2.1 Cell Culture and Drug Treatment
HeLa cells of human cervical carcinoma origin (ATCC catalogue number unknown) were
cultured in a 96-well plate at a seed density of 1x104
cells per well. A total of four wells were seeded,
each containing 100 µl of tissue culture medium [Dulbecco's Modified Eagle's medium (DMEM)
suspended with 10% fetal bovine serum (FBS)]. The seeded cells were cultured in an incubator at 37o
C
for 24 hours.
Etoposide and Staurosporine anti-cancer agents were dissolved in dimethyl sulphoxide
(DMSO), a drug delivery vehicle, to a stock concentration of 5 mM and 1 µM, respectively. After 24
hours of cell culture, we replaced the spent medium in each well with the appropriate drug treatment
and IncuCyteTM
Caspase 3/7 Reagent (CasR) as detailed in Table 1. The cells were then returned to the
incubator for an additional 24 hours of culture.
4. Table 1. Etoposide and Staurosporine drug treatment conditions for HeLa cells in each well of the 96-well plate.
Well
No.
Final Concentration
Volume added to each well (µl)
Etoposide
Stock – 5 mM
Staurosporine
Stock – 1 µM
CasR
Stock - 500 µM
DMSO
Stock - 30%
DMEM
1 10 nM Staurosporine
+ 5 µM CasR
1 1 98
2 50 µM Etoposide
+ 5 µM CasR
1 1 98
3 10 nM Staurosporine
+ 50 µM Etoposide
+ 5 µM CasR
1 1 1 97
4 0.3% DMSO
+ 5 µM CasR
1 1 98
2.2 Fluorescence Microscopy
After approximately 24 hours of incubation with the apoptosis assay reagent CasR and different
drug treatment conditions, the cells were viewed at 20x magnification under a fluorescence
microscope. As the DNA intercalating dye, NucViewTM
488, has an excitation/emission wavelength of
485/515 nm [13], an excitation bandpass filter with a range of 460-490 nm was used to specifically
visualize the green fluorescence emitted in the fluorescein isothiocyanate (FITC) channel of the
microscope.
We imaged four different regions per well with each region having had two different images
taken; a bright field and its corresponding green fluorescence image. Image processing and cell count
were performed with the assistance of the software, ImageJ.
3. Results
3.1 Apoptosis Induction Efficiency
The drug treated HeLa cells were imaged using microscope under both bright-field (BF) and
green fluorescence (GF) conditions. The number of live cells were enumerated under BF while
apoptotic cells were scored under GF conditions. The data have been summarized in Table 2.
5. Table 2. Etoposide and Staurosporine apoptosis induction efficiency in HeLa cell after approximately 24 hours treatment
duration.
Imaged
Regions
Well 1 Well 2 Well 3 Well 4
10 nM Staurosporine 50 µM Etoposide 10 nM Staurosporine
+ 50 µM Etoposide
0.3% DMSO
(Control)
GF BF GF BF GF BF GF BF
1 36 181 22 103 43 66 1 356
2 33 168 22 120 37 73 3 241
3 44 143 29 170 32 71 5 296
4 36 169 26 125 34 91 7 322
Average 37 165 25 130 37 73 4 304
Standard
Deviation (±)
5 16 3 29 5 11 3 49
Total 202 155 110 308
Apoptotic (%) 18.3 16.1 33.6 1.3
Live (%) 81.7 83.9 66.4 98.7
Our results suggest that both Etoposide and Staurosporine are capable of inducing apoptosis in
HeLa cells. Independently, Etoposide caused apoptosis in 16.1% of HeLa cells while Staurosporine had
a similar efficiency of 18.3%. However, a co-administered drug therapy with both Etoposide and
Staurosporine nearly doubled the efficiency up to 33.6%. As our DMSO control demonstrated a near-
negligible apoptosis induction efficiency of 1.3%, we can be confident that the apoptotic events were
strongly attributed to the drug treatments.
3.2 Cellular Morphology Changes
Apoptosis have been known to exhibit characteristic morphological changes distinct from
necrosis. Figure 1 demonstrates the morphology of HeLa cells undergoing apoptosis by Etoposide and
Staurosporine induction. In addition, the figure also reveals non-apoptotic cell having altered cellular
morphology by the two drugs.
6. Figure 1. Composite bright-field (BF) and green fluorescence (GF) images of different HeLa cell culture regions under
different drug treatment conditions. (A) 10 nM Staurosporine, (B) 50 µM Etoposide, (C) 10 nM Staurosporine + 50 µM
Etoposide and (D) 0.3% DMSO control. Black arrows represent apoptotic cells while white arrows represent non-apoptotic
HeLa cells.
As evident by the green fluorescence, Figure 1 proves that HeLa cells can be induced to
undergo apoptosis by either Etoposide or Staurosporine. In general, we observed apoptotic cells to have
their plasma membrane bleb together with their cytoplasm before shrinking into irregular-shaped
spheres (Figure 1C: black arrow). Regardless of singular or co-administered drug therapy, apoptotic
cells appeared to have shriveled up with a massive loss to their cytoplasmic volume, whereas their
nuclear size remained relatively unchanged and comparable to control cells in Figure 1D. In terms of
nuclear morphology however, Staurosporine-induced apoptotic cells displayed a homogeneously
fluorescing nucleus (Figure 1A), while Etoposide-induced apoptotic cells showed fragmented, and
sometimes lobular substructures fluorescing (Figure 1B: black arrow). In the co-administered drug
therapy, there was a mixture of both nuclear morphologies (Figure 1C).
Figure 1D reveals that 0.3% DMSO is not cytotoxic and does not significantly interfere with the
drug treatment outcome. In this control culture, cells were observed to proliferate in close proximity
with one another. Furthermore, despite growing in a monolayer, these squamous cells were observed to
contain a large volume, conferring them a 3D appearance.
In contrast, Figure 1A demonstrates that in addition to Staurosporine ability to induce apoptosis,
it also is capable of altering the morphology of non-apoptotic cells. These non-apoptotic cells appeared
flatter, elongated and disjointed from one another. Similarly, in Figure 1B, it can be seen that Etoposide
7. treatment causes non-apoptotic cells to be further apart from each other, but these cells appear to have
either become swollen or multinucleated (Figure 1B: white arrow). When both drugs were co-
administered, the morphological alterations to non-apoptotic cells resembled more to that of
Staurosporine treatment.
4. Discussion
4.1 Effectiveness of Etoposide and Staurosporine
In human cervical carcinoma, the cytochrome c/Apaf-1/caspase-9 proteolysis cascade is usually
inactive due to p53 dysfunctionality [4][5]. DNA-damaging drugs like Etoposide and non-specific
protein kinase inhibitors such as Staurosporine have been documented to induce apoptosis in HeLa
cells [7][8]. Interestingly, both drugs have been reported to execute apoptosis through the cytochrome
c/Apaf-1/caspase-9 activation [6][8]. Also, studies have shown the ability of Staurosporine to bypass
activation of caspase-9 and induce apoptosis [8][9]. One of such study with HeLa cells reported
activation of caspase-8 to be a post-mitochondrial event downstream of caspase-9 activation [8]. On the
other hand, in addition to TopoIIα inhibition during S phase, Etoposide has been found to cause cell
death by stimulating the trimerization of Fas Ligand and Receptor (FasL-FasR) complex or by inducing
mitotic catastrophe [6][7]. Overall, Etoposide and Staurosporine demonstrates a huge potential to be
p53-independent anti-cancer agents.
In our analysis, Etoposide alone induced apoptosis in 16.1% of HeLa cells while Staurosporine
managed to achieve 18.3% (Table 2). Co-administration of both drugs yielded an apoptosis induction
efficiency of up to 33.6%. This data validates our expectation that both drugs are capable of inducing
apoptosis in human cervical carcinoma cells. In addition, these results suggest that co-administration
may have an additive effect to the apoptosis induction efficiency. In a closely related study, researchers
have reported that Staurosporine is capable of potentiating Etoposide-induced apoptosis in HeLa cells
up to three-folds by acting on events downstream of DNA damage [14]. As our co-administered drug
treatment yielded a summative instead of a multiplicative apoptosis induction efficiency, we argue that
Staurosporine may not be completely potentiating Etoposide-induced apoptosis. Considering the ability
of Staurosporine to trigger pathways slightly different from Etoposide, we propose additional co-
administration experiments with a range of drug concentrations to elucidate if these agents are additive,
synergistic or both.
4.2 Cell Behaviour to Apoptosis Inducers
Etoposide and Staurosporine-induced HeLa cells were observed to exhibit signs of membrane
blebbing, cytoplasm shrinkage and chromatin condensation. Although cells of both drug treatments
demonstrated these classical apoptosis characteristics, they differ slightly in their nuclear morphology.
Chromatin condensation in Staurosporine treated cells was noted to be homogenous. However,
chromatin in Etoposide treated cells appeared fragmented, and occasionally had lobular substructures
(Figure 1B: black arrow) within their nucleus.
One possible explanation for this prominent karyorrhexis observed in Etoposide treated cells
may be the inhibition of TopoIIα which causes single- and double-strand DNA breaks in the S phase
[6][7]. Furthermore, if these cells possess a defective G2 checkpoint, cell death may be triggered by
mitotic catastrophe [7], providing a possible explanation for the lobular substructures observed.
However, these could just be “heterochromatin masses resembling defined clumps”, as described in
one study using thymocytes [15].
8. On the other hand, non-apoptotic cells were also observed to have morphological alterations by
these drugs. The appearance of flatter and elongated Staurosporine-treated non-apoptotic cells (Figure
1A) could be due to the progressive loss of cytoplasmic actin microfilament bundles [16]. Swollen or
multinucleated (Figure 1B: white arrow) Etoposide-treated non-apoptotic cells might have had their
cell-cycle arrested [7]. Researchers have reported that Etoposide treatment beyond 24 hours at a
specific dose is required to trigger apoptosis is these cell-cycle arrested cells [7].
On a separate note, although we have observed reduced numbers in our cell cultures containing
the drug treatments, there were little or no cellular debris detectable. This strongly suggests that HeLa
cells might have engulfed the apoptotic cell debris. HeLa cells of epithelial origins have been said to
have phagocytic activity [17]. However, this cellular behavior, including observations mentioned
earlier, must be proven with real-time microscopy.
4.3 p53 Tumour Suppressor in HeLa Cells
The etiology of human cervical cancer often arises from an infection with high-risk oncogenic
type 16 or 18 HPVs [4]. Historically, HeLa cells were first isolated from a patient afflicted with this
disease [2][3]. In these cells, viral oncoprotein E6 and E7 have been shown to knock-down the function
of p53 and pRb, respectively [4][18][19].
The p53 tumor suppressor protein has long been regarded as the “guardian of the genome” as it
senses and responds to DNA damage through a set of cellular stress kinases [6][20]. In addition, it has
also been acclaimed to be the “policeman of the oncogenes” with the help of a p53-stabilizing protein,
alternate reading frame (ARF) [20]. In HeLa cells, constitutive expression of high-risk HPV E6
oncoproteins knocks-down both roles of p53, conferring cells the ability to bypass cell cycle
regulations and evade apoptosis [4][5].
One would then question why would a cell lacking p53 function be able to undergo apoptosis as
observed in our experiment with anti-cancer agents, Etoposide and Staurosporine? It turns out that the
defective p53 function need not be rescued for apoptosis induction as proven by a growing number of
research using these drugs to induce apoptosis through p53-independent pathways [6][7][8][9].
Although our HeLa cells were claimed to be of human cervical cancer origin, it does not
necessarily indicate a p53 loss of function. Also, the use of Etoposide and Staurosporine to successfully
induce apoptosis in these cells do not provide information on the cellular status of p53 as both drugs
could act in a p53-dependent or independent manner. With the additional concerns of contaminated
HeLa cell lines [2] and the ability of chemotherapeutics to reactivate p53 functionality [21], we cannot
confidently report the status of our HeLa cell line unless molecular characterization have been
performed.
4.4 Drug Dosage and Exposure Time
Determining the appropriate lethal dose and exposure time of a drug to eliminate cancer cells
provides important information for potential clinical applications. There have been only a few studies
that utilized 10 nM Staurosporine and 50 µM Etoposide to evaluate apoptosis induction potential in
9. HeLa cells. Nevertheless, based on the existing knowledge, it is evident that HeLa cells respond
differently to varying drug dosage and exposure time.
In one study, researchers used a pulse of 50 µM Etoposide in HeLa cells for only three hours,
and that resulted in an apoptosis induction efficiency of approximately 20.0% at 48 hours, 60.0% at 72
hours and 100% at 96 hours post-treatment [7]. In our experiment, when we incubated HeLa cell with
50 µM Etoposide without removal of the drug for 24 hours, we obtained an apoptosis induction
efficiency of 16.1%, which is close to that of the previous study at 48 hours. This might indicate that
HeLa cells require prolonged exposure to Etoposide and sufficient time for the drug’s mechanism of
action to achieve maximal apoptosis induction efficiency.
In a separate study, 50 nM Staurosporine was used to induce apoptosis in HeLa cells. Apoptosis
induction efficiency progressed gradually from approximately 18.0% at six hours to 60.0% at 24 hours
of drug treatment [22]. At 10 nM Staurosporine, we achieved 18.1% apoptosis induction efficiency at
the 24-hour time point. This illustrates that Staurosporine drug dose is crucial in terms of HeLa cell
elimination rate. Unfortunately, in both cases, we cannot derive information regarding the drug’s
maximum efficiency as the treatment did not extend beyond 24 hours.
4.5 Personalized Polytherapy
Based on our data, the additive effect of both Etoposide and Staurosporine was observed to be
twice as effective as compared to when they were administered separately in HeLa cells. Although
Staurosporine is considered a potential anti-cancer agent, it possesses undesired toxic effects which
limits its clinical applications [10]. Hence, a polytherapy containing Etoposide and a reduced dose of
Staurosporine may prove beneficial to cervical cancer patients sensitive to Etoposide treatment or have
developed resistance to this conventional therapy.
On top of genotyping patients for personalized medicine, we propose conducting a cell culture
of the patient’s tumour tissue in vitro and subjecting the culture to various anti-cancer agents, inclusive
of Etoposide- Staurosporine polytherapy, to determine the most suitable treatment option for patients
based on the calculated risks versus benefits.
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