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1324 Current Medicinal Chemistry, 2015, 22, 1324-1334

A Simple and Reliable Approach for Assessing Anticancer Activity In Vitro

Miguel López-Lázaro*

Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain

Abstract: Cancer patients need better anticancer drugs, and medicinal chemistry can play a critical
role in the discovery of these drugs. For an efficient drug discovery process, chemists working on the
synthesis of potential anticancer agents need to use reliable screening methods. These methods should
not only detect the compounds with the highest therapeutic potential, but should also predict whether
such potential is high enough to deserve additional attention. Unfortunately, the current strategies for
assessing anticancer activity in vitro are unable to do this reliably. This review article analyzes these
strategies and describes an alternative screening approach. It is based on establishing suitable experi-
mental conditions to detect compounds that improve the selective cytotoxicity of the drugs used in
cancer therapy. This patient-oriented approach is easy to implement and may help medicinal chemists, and other research-
ers involved in cancer drug discovery, assess in vitro anticancer activity more reliably.
Keywords: Antitumor activity, cancer, cytotoxic activity, drug discovery, screening, selectivity.

1. INTRODUCTION Medicinal chemists involved in cancer drug discovery

typically follow a cytotoxic potency-based screening ap-
The chances of survival of people diagnosed with local-
proach: the lower the concentration required to kill cancer
ized tumors are relatively high. Unless the tumor is in a vital
cells the higher the potential for cancer therapy. If the most
organ or problematic location, surgery and radiation thera-
cytotoxic compound of the series improves the cytotoxic po-
pies can generally eliminate the tumor cells and cure the dis- tency of the anticancer drug used as control, the new com-
ease. These therapeutic modalities are not curative when
pound deserves additional attention [2-7]. Another common
cells from primary tumors have already metastasized to un-
screening strategy consists of assessing the ability of the
known locations. When this very common situation arises,
compounds to act on particular therapeutic targets. For exam-
oncologists need to administer drugs systemically to try to
ple, because DNA topoisomerases and tubulin are the targets
reach and kill the cancer cells. Unfortunately, the ability of
of well-established anticancer drugs, the most potent modula-
the existing anticancer drugs to eliminate the cancer cells in tors of these targets are considered to be the most promising
patients with metastasis is low, and these patients do not
compounds of their series [8-16]. Recently, compounds have
usually overcome the disease. For example, the five-year
been screened and selected based on their ability to target
relative survival rate for patients with distant metastasis is
molecular differences between cancer cells and healthy cells,
4% in lung cancer, 28% in prostate cancer, 24% in breast
e.g., particular protein kinases [17-22]. Several recent studies
cancer, 13% in colorectal cancer, 3% in liver cancer, 16% in
have also focused on the screening of small-molecule modu-
melanoma, 12% in renal cancer, 27% in ovarian cancer, 5% lators of microRNAs [23, 24]. Researchers usually combine
in bladder cancer, 4% in esophageal cancer, and 2% in pan-
cytotoxic potency-based approaches with target-based ap-
creatic cancer [1]. Many patients within these percentages
proaches to identify the best compounds of their series, and
eventually die of the disease despite surviving five years
use animal models to confirm the in vitro anticancer activity
after diagnosis.
of their selected compounds [8, 9, 11, 14, 16, 19, 20].
Because pharmacotherapy is the main form of treatment After analyzing the type of drugs that cancer patients
for patients with advanced metastatic cancers, it is vital to
need, this article shows that the current screening methods
discover better anticancer drugs. Medicinal chemistry plays
are unreliable to identify this type of drugs. Then, it de-
an important role in cancer drug discovery and development.
scribes a simple and patient-oriented screening approach,
For an efficient drug discovery process, chemists working on
which may help researchers assess the in vitro anticancer
the synthesis of potential anticancer agents need to use reli-
activity of their compounds more reliably.
able screening methods. These methods should not only de-
tect the compounds with the highest therapeutic potential,
2. CANCER PATIENTS NEED DRUGS THAT IM-
but should also predict whether such potential is high enough
PROVE THE ABILITY OF THE EXISTING DRUGS
to deserve additional attention.
TO KILL THEIR CANCER CELLS SELECTIVELY
*Address correspondence to this author at the Department of Pharmacology,
Faculty of Pharmacy, University of Seville, Spain; C/ Profesor Garcia Gon- When one treats cancer cells with standard anticancer
zalez 2, 41012, Sevilla, Spain; Tel: +34 954 55 63 48; drugs and examines the cells under the microscope, one ob-
Fax: + 34 954 55 60 74; E-mail: mlopezlazaro@us.es serves that specific concentrations and exposure times result

1875-533X/15 $58.00+.00 © 2015 Bentham Science Publishers

A Simple and Reliable Approach for Assessing Anticancer Activity Current Medicinal Chemistry, 2015, Vol. 22, No. 11 1325

in a 100% cancer cell death. Many of these drugs kill all the
cancer cells at micromolar concentrations, and others do it at
nanomolar or millimolar concentrations. Researchers have
also found numerous compounds not used in cancer therapy
despite their ability to kill cancer cells at very low concentra-
tions. These observations raise questions that chemists work-
ing on the synthesis of potential anticancer agents ask cancer
pharmacologists: If we already have drugs that are excellent
at killing cancer cells, why do these drugs not save the lives
of patients with metastatic cancers? Why do oncologists of-
ten use drugs that kill cancer cells at high concentrations
instead of compounds that do it at lower concentrations? At
what concentration should a new compound kill cancer cells
to have potential for cancer therapy?
The current drugs do not usually save the lives of patients
with metastatic cancers because they have a limited ability to
kill cancer cells at concentrations that do not significantly
affect healthy cells. This narrow selectivity implies that on-
cologists cannot use the doses of the drugs required to kill all
the cancer cells of their patients. If they used such doses,
they would also kill the cells from their normal tissues and
would cause the death of the patients. As a poor alternative,
oncologists have to use the maximum doses tolerated by the
patients, which are generally insufficient to reach the drug
concentrations required to eliminate all their cancer cells.
The purpose of this strategy is to eliminate as many cancer
cells as possible without killing the patient, usually with the
final aim of extending patient survival (Fig. 1). The answer
to the second question is that oncologists often use drugs that
kill cancer cells at high concentrations because the key fea-
ture of an effective anticancer drug is its ability to kill cancer
cells selectively and not its ability to kill cancer cells at low
concentrations. A drug that kills cancer cells at 1 mM with-
out affecting healthy cells at 100 mM will probably kill more
cancer cells in patients than a drug that kills both cell types
at 1 nM. The answer to the third question is that the new
compound should kill cancer cells at concentrations that do
not significantly affect nonmalignant cells in order to have
potential for cancer therapy [25].
Selectivity is the most important feature of an effective
anticancer drug. Understanding how much selectivity a new
drug should have to be clinically effective is essential. If
possible, the drug should kill all the cancer cells of the pa-
tients without significantly affecting their healthy cells. It
should match the selectivity of antibiotics, which can kill the
bacterial cells of our bodies at concentrations that do not
significantly affect our cells. An anticancer drug with this
selectivity would probably save the lives of patients with
metastatic cancers, like antibiotics commonly save the lives
of patients with bacterial infections. If this is not possible,
the new drug should at least improve the survival rates of
cancer patients treated with the current pharmacological

Fig. (1). The anticancer drug sorafenib has a limited ability to kill cancer cells selectively. Sorafenib is the standard of care for patients
with unresectable or metastatic hepatocellular carcinoma (liver cancer). This oral multikinase inhibitor is known to induce direct and indirect
cytotoxic effects against cancer cells. Skin toxicity is one of the most common dose-limiting side effects of sorafenib. The recommended
maximum tolerated dose for patients receiving sorafenib is 400 mg twice daily, which results in mean plasma concentrations of approxi-
mately 10 µM. In the experiments shown in this Figure, human liver hepatocellular carcinoma cells (HepG2) and human skin nonmalignant
cells (VH10) were exposed to sorafenib 10 µM and 100 µM for 24 h. Sorafenib was then eliminated and the cells were exposed to drug-free
medium for an additional period of 48 h. At this point, representative photographs were taken, and the percentage of cell viability (shown at
the right bottom of the photographs) relative to the untreated cells was estimated with the MTT assay. At high concentrations (100 µM),
sorafenib killed all the liver cancer cells, but also killed the nonmalignant skin cells. At the maximum concentrations tolerated by patients (10
µM), sorafenib showed some selectivity for the cancer cell line, but could not kill all the liver cancer cells. This limited selectivity probably
explains why most of the patients with hepatocellular carcinoma receiving sorafenib live several months longer than untreated patients but do
not usually overcome the disease (the five-year survival rate in patients diagnosed with advanced metastatic liver disease is approximately
3%) [1, 65-67]. Figure taken from reference [25].
1326 Current Medicinal Chemistry, 2015, Vol. 22, No. 11
therapies. A drug that improved the ability of the standard
drugs to kill cancer cells selectively would probably increase
patient survival. We should therefore look for drugs that im-
prove the selectivity of the existing anticancer drugs [25].
3. CURRENT SCREENING APPROACHES CANNOT
RELIABLY IDENTIFY THE TYPE OF DRUGS THAT
CANCER PATIENTS NEED
Cancer patients need drugs that improve the ability of the
standard drugs to kill their cancer cells without significantly
affecting their healthy cells. The most common screening
strategies cannot detect this type of drugs reliably.
Chemists involved in cancer drug discovery typically fol-
low a cytotoxic potency-based screening approach: a com-
pound is promising if it kills cancer cells at low concentra-
tions and improves the potency of the standard drugs. For
example, several compounds have recently been considered
promising anticancer agents based on their ability to kill can-
cer cells at lower concentrations than those of the standard
drugs etoposide [26], cytarabine (ara-C) [27], 5-fluorouracyl
[28], doxorubicin [29] and cisplatin [7, 30]. Despite its wi-
despread use, this approach is unsound and unreliable.
It is unsound because the aim of this approach is to find
something that we do not need. Cancer patients do not need
drugs that kill their cancer cells at low concentrations. As
explained previously, they need drugs that kill their cancer
cells at concentrations that do not significantly affect their
healthy cells. If a drug kills cancer cells at low concentra-
tions and also kills normal cells at similar concentrations, the
maximum doses tolerated by the patients will be insufficient
to reach the drug concentrations required to eliminate their
cancer cells. If a drug kills cancer cells without significantly
affecting nonmalignant cells, it does not matter much at what
concentrations it kills the cancer cells.
It is unreliable because the ability of a compound to kill
cancer cells at low concentrations is not related to its ability
to kill cancer cells selectively. For example, after reporting
that several isoprenyl-thiourea and urea derivatives killed
colon cancer cells at low concentrations and improved the
potency of the anticancer drug 5-fluorouracil, we found that
these compounds were equally cytotoxic towards nonmalig-
nant cells [31]. More recently, we assessed the anticancer
activity of a series of new aziridine derivatives, and found
that the most cytotoxic compound against a cancer cell line
did not show any selectivity versus a nonmalignant cell line
from the same tissue (it was actually 7 times more cytotoxic
against the normal cell line). We selected for further studies
a compound that was less cytotoxic in the cancer cell line,
but more selective. When we tested the cytotoxicity of this
compound against three pairs of human cancer cell lines and
nonmalignant cell lines, we observed that the selectivity of
this aziridine against breast cancer cells versus normal cells
from three different tissues was approximately 50-100 fold
[32]. These experimental data demonstrate that cytotoxic
potency and selective cytotoxicity are two unrelated parame-
ters. Therefore, when we show that our compounds kill can-
cer cells at low concentrations, we are not revealing whether
or not they have potential for cancer therapy.
Miguel López-Lázaro
The ability of a compound to modulate molecular targets
involved in cancer cell proliferation and survival can neither
reliably predict its selectivity towards cancer cells. For ex-
ample, the enzymes DNA topoisomerases are necessary for
cancer cell proliferation, and several topoisomerase inhibi-
tors are useful anticancer drugs. This does not mean, how-
ever, that a good inhibitor of these enzymes is a promising
anticancer compound. We have reported that curcumin is an
efficient inhibitor of DNA topoisomerases [33] with a very
limited selectivity towards cancer cells [34, 35]. Wang et al.
recently screened a chemical library of 359,484 compounds
to identify potential inhibitors of the steroid receptor coacti-
vators SRC-3 and SRC-1; these coactivators are involved in
cancer cell proliferation and survival. They identified the
cardiac glycoside bufalin as a potent inhibitor of SRC-3 and
SRC-1. This compound also inhibited breast cancer cell
growth at nanomolar concentrations and reduced tumor
growth in mice xenotransplanted with breast cancer cells.
Based on these observations, the authors discussed that bu-
falin could be useful for cancer therapy [36]. Clifford and
Kaplan observed, however, that human breast nonmalignant
cells were more sensitive than human breast cancer cells to
the cytotoxic activity of bufalin [37]. This suggests that bu-
falin would probably be ineffective in breast cancer therapy
[38].
Researchers do not routinely use healthy cells for assess-
ing anticancer activity in vitro because they may not think it
is necessary. In the end, no matter what screening approach
we follow, we will have to use animal models to assess pre-
clinical anticancer activity properly. These models will con-
firm the anticancer activity of the selected compounds, and
will also reveal if they induce toxicity against healthy tis-
sues.
Even if performed properly, in vivo animal experiments
cannot compensate for a deficient screening approach. First,
although animal models can show if the chosen compounds
induce selective anticancer activity in vivo, they cannot re-
veal the anticancer activity of the compounds left behind in
the screening process. Assessing selectivity after the screen-
ing step is an inadequate approach to identify the most selec-
tive compound of a chemical series. If we accept that selec-
tivity is the key feature of an effective anticancer drug, we
should assess selectivity as part of, and not after, the screen-
ing process so that we can detect the best anticancer com-
pounds of our series. Second, rodent models can show the
selectivity of a compound in human cancer cells versus ro-
dent normal cells (xenograft models), or in rodent cancer
cells versus rodent normal cells (allograft and spontaneous
models). However, animal models cannot show if a com-
pound kills human cancer cells versus human normal cells
selectively. This distinction is important to avoid experimen-
tal artifacts caused by species differences in sensitivity. For
example, we have shown that rodent cells are extremely re-
sistant (over 1000-fold) to the cytotoxicity of some cardiac
glycosides in relation to human cancer and nonmalignant
cells [39]. This means that if we assess the anticancer activ-
ity of these compounds in mice xenotransplanted with hu-
man cancer cells, we will find a marked in vivo anticancer
activity [36, 40-43]. This activity, however, is probably
caused by the ability of cardiac glycosides to selectively kill
human cells versus rodent cells rather than by their ability to
A Simple and Reliable Approach for Assessing Anticancer Activity
selectively kill human cancer cells versus human healthy
cells [39, 44, 45]. This can explain why the cardiac glycoside
bufalin inhibited tumor growth in mice xenografted with
human breast cancer cells [36] despite its lack of selectivity
[38]. These observations suggest that animal models cannot
substitute a proper in vitro design.
An inadequate in vitro design may result in the selection
of useless compounds and in the failure to detect promising
anticancer agents. In the first case, we will waste resources
trying to prove in animal models, and too many times in can-
cer patients, the anticancer activity of ineffective com-
pounds. In fact, most of the compounds selected for further
studies based on the current screening strategies do not show
relevant anticancer effects when tested in animal models of
metastasis and in clinical trials [46-48]. Drug attrition rates
for cancer are actually much higher than in other therapeutic
areas, and only 5% of compounds that enter clinical trials are
licensed after demonstrating sufficient efficacy in phase III
testing [47]. But the most worrying consequence of an in-
adequate in vitro screening is that we are probably throwing
away useful anticancer drugs. Thousands of novel com-
pounds are routinely discarded for not being the most cyto-
toxic of their series or the best modulators of particular mo-
lecular targets. Since these drug features are poor predictors
of therapeutic potential, an unknown number of these com-
pounds might be better than the existing anticancer drugs.
Finally, let us consider the following situation to examine
the ability of the current screening strategies to detect effec-
tive anticancer drugs reliably. Let us imagine that one of us
is diagnosed with advanced metastatic liver disease. Like
most of the patients in this situation, we would only survive
several months after diagnosis despite being treated with
novel anticancer drugs such as sorafenib (see Fig. 1 legend).
Let us imagine that we have a collection of 1000 compounds,
and that one of them is an effective cure for patients with our
cancer. Could we identify this effective anticancer com-
pound using the current in vitro approaches?
Some researchers would screen the collection against
liver cancer cells and would select the most cytotoxic com-
pound. Others would select the best modulator of a particular
therapeutic target. For example, we could choose the best
inhibitor of a specific protein kinase of a particular signal
transduction pathway involved in liver cancer cell prolifera-
tion and survival. We could also opt for a more classic thera-
peutic target and select, for instance, the best topoisomerase
inhibitor or antitubulin compound. We could also combine
several of these screening strategies. It is easy to predict that
the most cytotoxic compound would not be the best topoi-
somerase inhibitor, the best antitubulin compound, and the
best inhibitor of all the molecular targets of the multiple sig-
nal transduction pathways involved in liver cancer cell pro-
liferation and survival.
What would we think if a problem only has one solution
and each of the methods used to solve it leads to different
solutions? We would probably think that only one of the
methods is adequate, or that all of them are inadequate. If we
think that only one is adequate, which one would it be?
Would it be the one based on assessing cytotoxic potency or
the one based on assessing mechanisms of action? If we
think that the second option is the most suitable one, which
Current Medicinal Chemistry, 2015, Vol. 22, No. 11 1327
of the many potential mechanisms of action of the active
compound would we select for our screening protocol? What
if the anticancer drug that we are trying to identify acted on a
molecular target not yet discovered? This example suggests
that the most common screening approaches do not reliably
detect the best anticancer compound in a group of potential
anticancer agents. It also suggests that we need to develop
novel strategies to assess in vitro anticancer activity more
reliably [25].
4. A SIMPLE APPROACH FOR ASSESSING ANTI-
CANCER ACTIVITY IN VITRO
Cancer patients need drugs that improve the ability of the
existing drugs to kill their cancer cells without significantly
affecting their healthy cells. This patient-oriented approach
aims to establish the most adequate experimental conditions
to answer the following question: Can any of my compounds
improve the ability of the current drugs to kill cancer cells
without affecting nonmalignant cells from suitable tissues?
The general approach consists of 1) exposing cancer cells
and nonmalignant cells to the experimental drugs and to the
standard anticancer drugs, 2) estimating cell viability with a
simple cytotoxicity test, 3) calculating one or several cyto-
toxicity parameters (e.g., IC50 values) for each drug in each
cell type, 4) calculating a selectivity index for each drug
(e.g., dividing the IC50 value in the nonmalignant cells by
that in the cancer cells) and 5) comparing the selectivity in-
dices of the experimental drugs with those of the standard
anticancer drugs [25, 49, 50]. Several aspects need to be con-
sidered for implementing this approach.
4.1. Selection of Cancer Cells, Nonmalignant Cells and
Standard Anticancer Drugs
The first important aspect to consider for implementing
this approach is the selection of the cancer cells and nonma-
lignant cells. Distinguishing between cancer cells and nonma-
lignant cells and being familiar with several basic terms and
concepts is important for this purpose. Unlike nonmalignant
cells, cancer cells proliferate and disseminate in an uncon-
trolled manner and are able to induce tumors following inocu-
lation into susceptible animals. Primary cells are cells taken
directly from organisms, and can be regarded as such until
they are successfully subcultured for the first time. Cell lines
are cells derived from organisms after the first successful
subculture. The term nonmalignant cells can be used for pri-
mary nonmalignant cells, for cell lines derived from primary
nonmalignant cells that may have been altered to facilitate
cell growth in culture (e.g., cells that have been immortalized
through transfection of the catalytic component of the enzyme
telomerase reverse transcriptase hTERT), or for fetal cells.
The terms normal cells and healthy cells commonly refer to
non-diseased cells and, therefore, to nonmalignant cells.
Ideally, we should use primary cancer cells and primary
nonmalignant cells for implementing this approach. Because
primary cells are freshly derived from living organisms, they
mimic the physiological state of cells in vivo more closely
than cell lines, which may develop changes (e.g., unstable
karyotypes) as a result of subculturing. However, primary
cells are less convenient to use than cell lines; one needs at
least one subject to obtain the primary cells required for each
1328 Current Medicinal Chemistry, 2015, Vol. 22, No. 11
independent experiment. Their value in the laboratory setting
is limited, especially when large quantities of cells are re-
quired. Well-characterized cancer and nonmalignant cell
lines are suitable for implementing this approach.
Both the cancer cells and the nonmalignant cells should
be human to avoid species differences in sensitivity. For ex-
ample, as discussed previously, rodent cells are extremely
resistant to the cytotoxicity of cardiac glycosides. Therefore,
if we use a human cancer cell line and a rodent nonmalignant
cell line to evaluate the selectivity of a cardiac glycoside, we
will mistakenly find a selective anticancer effect caused by
species differences in sensitivity. The cancer cells and non-
malignant cells should also be from the same tissues to avoid
tissue differences in sensitivity. Imagine a compound that
kills cancer and normal cells from the lungs at 1 µM and
cancer and normal cells from the skin at 100 µM. If we as-
sess the selectivity of this compound using lung cancer cells
versus skin normal cells, we will find a selective anticancer
effect caused by tissue differences in sensitivity.
Another important aspect to consider is the number of
cell lines to use. Ideally, we should assemble a panel of well-
characterized cancer cell lines representative of all cancer
types, and a variety of nonmalignant cells. The normal cells
should be from the same tissues than those of the cancer cells
and from tissues commonly affected by cancer pharma-
cotherapy. With this panel of cell lines, we would reliably
predict whether or not any of the compounds has potential
for cancer therapy. If we use fewer cancer cell lines, useful
compounds for specific types of cancer may be missed. If we
use fewer nonmalignant cell lines, we may miss toxicity to-
wards particular tissues that could limit the therapeutic po-
tential of the compounds. Despite these limitations, we can
assess anticancer activity reliably using a much lower num-
ber of cell lines.
If the study is focused on a particular cancer, the mini-
mum requirement for implementing this screening approach
is to use a human cancer cell line, a human nonmalignant
cell line from the same tissue, and an anticancer drug used to
treat patients with the selected cancer. This simple approach
is suitable to screen rapidly the in vitro anticancer activity of
large series of compounds against a particular cancer. If none
of the compounds improves the selectivity index of the stan-
dard anticancer drugs, the compounds have a little potential
for the treatment of the selected cancer [51]. If one of the
compounds improves the selectivity index of the standard
drugs, the compound is promising; however, we should use
more nonmalignant cell lines to assess anticancer activity
more robustly. Using more normal cell types is essential to
detect possible toxicity to normal tissues that could limit the
potential use of the new compound. For example, a com-
pound that kills lung cancer cells at 1 µM and lung nonma-
lignant cells at 100 µM will not be clinically effective if it
also kills normal cells from other tissues (e.g., skin cells or
blood cells) at 1 µM. If the study is focused on one cancer, in
vitro anticancer activity can be assessed robustly with sev-
eral cancer cell lines representative of the most common
cancer subtypes and with several nonmalignant cell lines
originated from a variety of normal tissues. This little panel
of cell lines could be used to confirm the activity of com-
pounds passing the initial screening step.
Miguel López-Lázaro
If the study is not focused on a particular cancer, we
could use several pairs of cancer cell lines and nonmalignant
cell lines, as well as standard drugs used to treat patients
with the selected cancers. It is important to note that specific
anticancer drugs are used only in particular cancers. There-
fore, we should calculate selectivity indices for the experi-
mental compounds in each cancer and compare them with
the selectivity indices of the standard drugs used in those
cancers. For example, imagine that we decide to screen a
series of compounds against cancer and healthy cells from
the colon, lungs and liver, and that we use the standard anti-
cancer drugs 5-fluorouracil, cisplatin and sorafenib. To show
that one of our compounds could be useful for treating pa-
tients with colon cancer, we should use the IC50 values calcu-
lated for our compound and for the standard drug 5-
fluorouracil in the colon cancer cell line and in the three
nonmalignant cell lines (i.e., colon, lung and liver). Then, we
should calculate a selectivity index for our compound and for
5-fluorouracil dividing the mean IC50 value in the three
nonmalignant cell lines by the IC50 value in the colon cancer
cell line. Finally, we should see if the selectivity index of our
compound is higher than that of 5-fluorouracil.
Depending on the aim of the study, we can use particular
types of cancer cells. If the experimental treatment is being
developed for specific patients, primary cells from these pa-
tients could be used to better predict the effectiveness of the
treatment or to avoid the administration of inactive che-
motherapeutics to them [52, 53]. Treatment failure has been
associated with the ability of cancer cells to develop drug
resistance. We, therefore, could also use resistant cancer cells
for implementing this approach. Evidence also suggests that
resistance to treatment may be due to the fact that current
treatments preferentially target non-stem cancer cells. Cancer
stem cells have been associated with resistance to treatment,
worse prognosis and recurrence of the disease. Another way
that could help to identify potential anticancer drugs would be
to evaluate their effects on cancer stem cells [54-56].
4.2. Cell Treatment Conditions
Cell density is an important parameter to consider to ob-
tain reliable results. The cytotoxicity of a drug in cells grown
at low density may be different from that in cells grown at
high density. For example, the IC50 values for 5-fluorouracil
and a novel merosesquiterpene were approximately 6 times
lower in MCF7 breast cancer cells seeded at 5 x103 cells/cm2
than in the same cells seeded at 5 x104 cells/cm2 [57]. Be-
cause nonmalignant cells generally grow slower than cancer
cells, they are often seeded at higher densities than cancer
cells, which may originate unreal selectivity values. The
screening approach discussed in this manuscript minimizes
these false positives. Provided that the density at which we
seed each cell line is the same for the experimental drugs and
the standard drugs, we can seed different cell lines at differ-
ent densities. What matters in this approach is whether or not
the experimental compounds improve the selectivity of the
current anticancer drugs and not the magnitude of their IC50
values or selectivity indices in particular cell types.
The compounds should be tested at concentrations that
allow the determination of the cytotoxic parameters required
to calculate the selectivity indices. For example, if we show
A Simple and Reliable Approach for Assessing Anticancer Activity
that the IC50 value of a compound is e.g. 50 µM in cancer
cells and >100 µM in nonmalignant cells, we are not truly
revealing its anticancer potential. We are simply showing
that its selectivity index is higher than 2, which could be 3
(low therapeutic potential) or 10000 (high therapeutic poten-
tial). When the solubility of the compounds is not a problem,
we should use wide concentration ranges to allow the calcu-
lation of the required cytotoxic parameters in all the cell
lines.
Drug exposure times and drug-free recovery periods may
change the cytotoxic potency and selectivity of particular
compounds. Some drugs need more time to kill cells than
other drugs. If we use short exposure times and recovery
periods, we may underestimate the cytotoxicity of drugs in-
ducing a slow cytotoxic effect. Other compounds may trigger
selective cytotoxic effects at short exposure times and non-
selective cytotoxic effects at long exposure times. The selec-
tivity of these compounds could be missed if cell viability is
assessed after a long drug incubation period (e.g., 72 h), but
could be detected after a shorter drug exposure time (e.g., 4
h) followed by a recovery period (e.g., 68 h). In addition, it
should be noted that many anticancer drugs used in patients
have a very short biological half life (time required to elimi-
nate 50% of the drug from the plasma). Short drug exposure
times followed by drug-free recovery periods may therefore
mimic the in vivo activity of these drugs more closely than
long drug exposure times. Drug exposure times and drug-
free recovery periods should be designed flexibly. However,
if we test the experimental compound and the standard drug
following several treatment schedules, we should not only
compare the selectivity indices obtained under the same
schedules, but also under the most selective schedules.
4.3. Methods for Assessing Cell Viability
Once the cells have been exposed to the experimental
compounds and the standard anticancer drugs, we need to
estimate how many viable cells remain at the end of the ex-
periments. This section does not seek to review all the avail-
able assays used to estimate cell viability in culture. It briefly
discusses several standard assays that can be used to imple-
ment the screening approach shown in this article. Each as-
say has its own set of advantages and disadvantages, and the
selection of a particular method depends on many factors
such as the number of samples to screen, its suitability for
high-throughput screening (HTS), or the infrastructure and
research funds of the laboratory. The assays discussed next
can be used in multi-well formats, and have been reviewed
or described in detail elsewhere [58-63]. References [58]
(freely available at http://www.ncbi.nlm.nih.gov/books/
NBK144065/) and [63] describe in detail several common
protocols for assessing cell viability.
The MTT assay is an inexpensive and widely used col-
orimetric assay. It was the first convenient 96-well method
developed for screening large numbers of samples. The MTT
assay is based on the ability of viable cells to convert the
MTT compound (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) into an insoluble and purple
formazan product; dead cells are metabolically inactive and
cannot reduce the MTT into the colored product. After an
incubation period (generally 1-4 h) of the cells with the MTT
Current Medicinal Chemistry, 2015, Vol. 22, No. 11 1329
and a solubilization step, the quantity of the colored product
is measured at 570 nm using a plate reading spectrophotome-
ter. Like all assays that rely on an active cellular metabolism,
any change in culture conditions that alters the metabolism
of the cells (e.g., high cell confluence, changes in pH, deple-
tion of essential nutrients) will likely affect the rate of MTT
reduction into the formazan product and will lead to a loss of
linearity between absorbance and cell number. In addition,
like all assays that rely on a reduction reaction, the MTT is
subject to interferences from compounds with inherent re-
ductive capacity (e.g., ascorbic acid and sulfhydryl-
containing compounds such as reduced glutathione). At high
concentrations and exposure times, the compound MTT in-
duces cytotoxic effects. The solubilization step required for
the MTT assay can be eliminated using newer tetrazolium
reagents (e.g. XTT) that are reduced by viable cells into
soluble formazan products. The assays based on these newer
tetrazolium reagents also have their advantages and disad-
vantages [58-60].
The resazurin assay is a redox-based colorimetric or
fluorometric assay, which employs a methodology similar to
that of the tetrazolium assays (MTT, XTT). This method
relies on the capacity of viable cells to reduce the blue com-
pound resazurin into the pink, fluorescent and soluble prod-
uct resorufin. The quantity of resorufin produced is propor-
tional to the number of viable cells. Fluorescence can be
quantified using a fluorometer equipped with a 560 nm exci-
tation / 590 nm emission filter set. Although resorufin can
also be quantified by measuring a change in absorbance,
spectrophotometric determination is not often used because
of its much lower sensitivity. When used as a fluorometric
assay, its sensitivity is higher than that of the tetrazolium
assays. However, the resazurin assay is also subject to
chemical interferences caused by reducing compounds. Re-
sazurin is also cytotoxic at high concentrations and exposure
times. Fluorescent compounds may also interfere with the
resorufin product. An important advantage is that resazurin
reagents probably generate the most cost-effective data on a
per well basis [58-60].
The sulforhodamine B (SRB) assay is a cost-effective
and widely used colorimetric method for screening, in which
cell density determination is based on the measurement of
cellular protein content. After drug treatments, cells are fixed
with trichloroacetic acid and stained with the SRB com-
pound. The excess dye is then removed by washing the
plates with acetic acid, and the protein-bound dye is dis-
solved in Tris base solution for spectrophotometric determi-
nation at 510 nm. Its sensitivity is comparable to that of
some fluorometric methods. Another important advantage is
that SRB staining is independent of cell metabolic activity;
therefore, fewer steps are required to optimize assay condi-
tions for cell lines with different metabolic activities. In ad-
dition, the SRB assay is not subject to chemical interferences
caused by reducing compounds. The SRB method, however,
does not distinguish between viable and dead cells, although
this does not seem to compromise its ability to detect cyto-
toxic effects of a drug. Several studies have shown that re-
sults from the SRB assay correlate well with those of the
MTT assay, although the IC50 values of compounds tested
using the SRB method generally are slightly higher. The
SRB assay is the screening method used by the National
1330 Current Medicinal Chemistry, 2015, Vol. 22, No. 11
Cancer Institute (NCI) in their anticancer drug discovery
program [62, 63].
The ATP assay is a fast and highly sensitive lumines-
cence method widely used for estimating cell viability in
high-throughput screening. Cells contain regulated levels of
ATP, and drug-induced cytotoxicity results in a loss of abil-
ity to synthesize new ATP along with a rapid depletion of
cellular ATP by endogenous ATPases. ATP is quantified by
measuring the light produced through its reaction with the
firefly enzyme luciferase using a luminometer. The amount
of light produced is directly proportional to the number of
viable cells. Commercial kits contain a detergent to lyse the
cells, ATPase inhibitors to stabilize the ATP that is released
from the lysed cells, luciferin as a substrate, and luciferase to
catalyze the reaction. The main advantage of this assay is its
high sensitivity, which enables miniaturization into 1536
well formats for HTS protocols. In addition, luminescence
signals are not altered by intrinsically fluorescent test com-
pounds. Another advantage of this assay is that, like the SRB
assay and unlike the tetrazolium and resazurin assays, it does
not require an incubation step of the substrate with a popula-
tion of viable cells to yield the product that generates the
signal, which eliminates the step of returning the cells to the
incubator. However, the assay would detect possible changes
in cellular ATP levels not related to changes in cell viability.
In addition, the luciferase reaction is susceptible to tempera-
ture changes and may also be altered by luciferase inhibitors
[58-60].
The lactate dehydrogenase (LDH) assay has been widely
used to detect in vitro cytotoxicity. LDH is a cytosolic en-
zyme that is released into cell culture media when the plasma
membrane is damaged. The released LDH can be quantified
by a coupled enzymatic reaction. First, LDH catalyzes the
conversion of lactate to pyruvate via reduction of NAD+ to
NADH. Second, diaphorase uses NADH to reduce an appro-
priate redox reagent such as a tetrazolium compound or resa-
zurin into a colored formazan or fluorescent product. The
levels of this product is directly proportional to the amount
of released LDH in the medium and, therefore, to the amount
of dead cells. Although LDH activity can be measured spec-
trophotometrically, fluorescence detection is advisable to
increase sensitivity. This assay is susceptible to background
signal from serum sources of LDH found in supplemented
growth media, which elevate the background signal. This
method is also subject to assay artifacts by compounds that
inhibit LDH activity and to fluorescence or color interfer-
ences. An important inconvenient is that activity-based sur-
rogates of cell death such as LDH have a finite enzymatic
half-life. The loss of LDH activity over time can lead to un-
derestimation of cytotoxicity in cells exposed for relatively
long incubation periods (48-72 h) to drugs (or drug concen-
trations) that induce a fast cytotoxic effect [59].
Although these assays are based on different concepts,
there is usually a good correlation between the results ob-
tained with different assays. For example, although the MTT
assay measures cellular activity and the SRB assay measures
cellular content, comparisons of data from several hundred
compounds screened in parallel by MTT and SRB assays
yielded quite similar results [63]. The possible experimental
artifacts caused by a particular assay could be prevented and
Miguel López-Lázaro
detected using two independent assays [59, 60]. Observing
the cells under the microscope is also a good practice to de-
tect these possible artifacts, particularly when the number of
test compounds is small. If we use a particular assay, and the
results do not faithfully represent what we see under the mi-
croscope (cell number and morphology), we should consider
repeating the experiments using another assay.
4.4. Expression and Discussion of Results
After estimating cell viability with a cytotoxicity assay,
we need to calculate one or several cytotoxicity parameters
(e.g., IC50, IC90, LC50) for each drug in each cell type. Then,
we should use these cytotoxicity parameters to calculate one
or several selectivity indices for each drug (e.g., dividing the
IC50 value in the nonmalignant cells by that in the cancer
cells). Finally, we should compare the selectivity indices of
the experimental drugs with those of the standard anticancer
drugs.
Selectivity indices based on only one cytotoxic parameter
(e.g., IC50) may be misleading when dose-response curves are
not analyzed carefully. Fig. (2) represents dose-response
curves of three hypothetical compounds in a cancer cell line
and a nonmalignant cell line. Each panel also shows two se-
lectivity indices based on IC50 values and IC90 values. In Fig.
(2A), the selectivity index based on the IC50 values represents
faithfully the selectivity of the compound observed in the
dose-response curves. In Fig. (2B and 2C), however, the se-
lectivity index based on the IC50 values may lead to misinter-
pretation of results. Dose-response curves in Fig. (2B) show
that the selectivity of the compound is very limited; however,
the selectivity index based on the IC50 values (171,3) suggests
that the compound has a high selectivity towards the cancer
cells. In Fig. (2C), the selectivity index based on the IC50 val-
ues (3,0) suggests that the compound has a limited selectivity
towards the cancer cell line. However, the dose-response
curves show that this compound kills all the cancer cells at
0.1 µM and all the healthy cells at 100 µM. This compound
could be useful if the reduced cell viability (40%) observed in
the normal cells in the 0.1-10 µM range is caused by reversi-
ble inhibition of cell proliferation. False positives (Fig. 2B)
and false negatives (Fig. 2C) obtained when selectivity indi-
ces are calculated based on only one cytotoxic parameter
(e.g., IC50) could be prevented by using additional selectivity
indices based on other cytotoxic parameters (e.g., IC90) or by
showing the dose-response curves.
In addition to comparing the selectivity indices of the ex-
perimental compounds with those of the standard anticancer
drugs, it is important to observe that none of the healthy cell
lines is highly sensitive to the cytotoxicity of the experimen-
tal compounds. We should not forget that a high toxicity in
normal cells from just one particular tissue may cause that
the maximum doses tolerated by the patients are insufficient
to reach the drug concentrations required to eliminate their
cancer cells.
4.5. Final Considerations
The screening approach discussed throughout this article
has the typical limitations of all in vitro methods. Because in
vitro conditions cannot represent in vivo conditions faith-
fully, compounds that work in vitro may not work in vivo
A Simple and Reliable Approach for Assessing Anticancer Activity Current Medicinal Chemistry, 2015, Vol. 22, No. 11 1331

A SI (IC50) = 141,3 B SI (IC50) = 171,3

SI (IC90) = 120,1 SI (IC90) = 1,2

100 healthy cells 100 healthy cells

90 cancer cells 90
cancer cells
80 80

% cell viability
% cell viability

70 70
60 60
50 IC50 50 IC50
40 40
30 30
20 20
10 IC90 10 IC90
0 0
0 0,01 0,1 1 10 100 0 0,01 0,1 1 10 100

concentration (µM) concentration (µM)

C SI (IC50) = 3,0
SI (IC90) = 764,1

100 healthy cells

90 cancer cells
80
% cell viability

70
60
50 IC50
40
30
20
10 IC90
0
0 0,01 0,1 1 10 100

concentration (µM)

Fig. (2). Selectivity indices based on IC50 values do not always represent selective anticancer activity faithfully. In panel A, the selectiv-
ity index based on IC50 values, SI (IC50), represents the selectivity of the hypothetical compound faithfully. In panels B and C, however, this
index does not truly represent what dose-response curves show. Graph B shows that the compound has a very limited selectivity towards the
cancer cells, and the SI (IC50) indicates a high selectivity (false positive). Graph C suggests that the compound can kill cancer cells selec-
tively, and the SI (IC50) indicates a very low selectivity towards cancer cells (false negative). Misinterpretation of results could be prevented
by showing the dose-response curves or by calculating an additional selectivity index, e.g., SI (IC90). IC50 values (concentration of compound
required to inhibit cell viability by 50%) and IC90 values (concentration of compound required to inhibit cell viability by 90%) for each com-
pound are not shown for simplicity. Selectivity indices (SI) are calculated by dividing the IC50 (or IC90) values in the healthy cells by those in
the cancer cells. See text for further details.

and vice versa. Another limitation is that we cannot exclude of tumor volumes), or do not test the experimental drugs
toxicity to all types of normal tissues by using a variety of and the standard drugs under comparable experimental
nonmalignant cells. Therefore, animal models are essential to conditions [25, 46].
assess preclinical anticancer activity properly. In addition
The screening strategy discussed in this article is based
to detecting pharmacokinetic limitations of the experimen-
on assessing selectivity. Evaluating selectivity is not a
tal compounds, animal models are important to confirm or
novel idea in anticancer drug discovery. Researchers from
detect possible in vivo activity and toxicity. To assess in
many laboratories use healthy cells to assess if active con-
vivo anticancer activity robustly, we should evaluate if the centrations of their selected compounds induce toxicity
experimental drug improves the survival rates of the stan-
against these cells. Although this strategy provides useful
dard drugs when tested under equivalent experimental con-
information about the selected compounds, it is inadequate
ditions in animal models representative of the patients who
to identify the most selective compound in a group of po-
would eventually receive the drugs (e.g., animal models of
tential anticancer agents. This strategy is also inadequate to
metastasis). Unfortunately, most in vivo studies do not use
show if the chosen compound improves the selectivity of
animal models of metastasis, do not assess survival as the the drugs used in cancer patients. Another negative aspect
endpoint of the experiments (they typically assess reduction
1332 Current Medicinal Chemistry, 2015, Vol. 22, No. 11
of assessing selectivity this way is that the magnitude of
this parameter changes much depending on the type of can-
cer cells and healthy cells that we choose; this makes inter-
pretation and extrapolation of results problematic. This
problem is minimized in the screening approach described
in this article. What matters in the screening approach dis-
cussed here is if any of the compounds improves the selec-
tivity of the drugs used to treat cancer patients, and not the
magnitude of their cytotoxic effects in particular cancer
cells and healthy cells.
This approach can be used for high-throughput screening
[50]. Current high-throughput screening approaches are typi-
cally based on testing library compounds at one concentra-
tion against one specific target. The same chemical libraries
are continuously screened against different anticancer tar-
gets. The approach discussed in this manuscript requires
each compound to be tested at several concentrations against
several cell types. This would require changes in the imple-
mentation and organization of the drug discovery process
and would increase costs. However, the cost of screening the
same libraries many times and of assessing the in vivo anti-
cancer activity of inefficient compounds is probably higher.
On top of this, the implementation and organization of the
drug discovery process should not have priority over solid
scientific foundations [64]. The only way forward to make
an impact in the lives of cancer patients when their tumor
cells have spread to unknown locations is to find drugs that
improve the ability of the current drugs to kill these cells
selectively. High-throughput screening approaches should be
designed to detect this type of drugs [50].
CONCLUSION
Cancer patients need drugs that improve the ability of the
existing pharmacological treatments to kill their cancer cells
without significantly affecting their normal cells. Instead of
looking for this type of drugs, the current screening strate-
gies typically look for drugs that kill cancer cells at low con-
centrations or that modulate particular targets involved in
cancer cell proliferation and survival. Because cytotoxic po-
tency and ability to modulate these targets do not reliably
predict selectivity towards cancer cells, the current screening
approaches result in the selection of ineffective compounds
and probably in the failure to detect promising anticancer
drugs. In the first case, researchers waste resources trying to
prove in animal models and in cancer patients the anticancer
activity of ineffective compounds. In the second case, re-
searchers discard compounds that could change the lives of
cancer patients.
This article describes an alternative screening approach.
It is based on establishing the most adequate conditions to
answer the question: Can the experimental compounds im-
prove the ability of the existing anticancer drugs to kill can-
cer cells without significantly affecting nonmalignant cells?
This patient-oriented approach is easy to implement and can
help researchers assess in vitro anticancer activity more re-
liably.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
Miguel López-Lázaro
ACKNOWLEDGEMENTS
I thank my research group for helpful discussions.
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