The Interaction of Structural Analogues of Phenothiazines in p53-Dependent Cellular Signaling Pathways.

1
Introduction
The functioning of the
human body is a very dynamic process, in
which cells constantly die and new ones are created. Unfortunately,
during this phenomenon, cells can also undergo mutations, which is
the basis of cancer development. More than half of human cancers contain
many different mutations of the TP53 gene encoding the p53 protein.
Its basic tasks in physiological conditions include maintaining genetic
stability. It also plays an important role in eliminating abnormalities
in the cell, by activating DNA repair mechanisms.
−

It is a barrier
against the initiation and progression of cancer. Its tasks include,
among others: repairing genetic material
,
regulating
the cell cycle, participating in apoptosis
,
and cell aging.

Mutations in the
TP53 gene cause poor prognosis in various types
of cancer. These are usually somatic changes that occur in cells during
carcinogenesis. The main effect of TP53
mutations in cancer cells is the loss of tumor suppressor properties,
which requires therapeutic reactivation of the protein. All mutations
occurring in the p53-dependent pathway can enable cancer development,
and restoring normal p53 protein functions enables inhibition of tumor
growth.

In colorectal cancer, p53
exists as wild-type or mutant, guiding
therapeutic approaches to prevent degradation or suppress dysfunction. Its activity is tightly regulated by interactions
with proteins like MDM2, which negatively control its stability and
function (Scheme
). Blocking these negative regulators in tumors
is crucial because MDM2 ubiquitinates p53, which leads to its degradation. Under normal conditions, MDM2 binds to p53,
catalyzing its ubiquitination and degradation in the proteasome, which
keeps p53 levels low and ensures proper cellular function.
,
There is a negative feedback loop between MDM2 and p53p53
activates the transcription of the MDM2 gene, and MDM2 limits the
activity of p53, which helps maintain balance in the cell.
−

In response to DNA damage, ATM/ATR kinases phosphorylate p53, preventing
its interaction with MDM2 and stabilizing the protein. Stable p53
translocates to the nucleus, activating transcription of genes responsible
for cell cycle arrest (e.g., p21), DNA repair (e.g., GADD45), and
apoptosis (e.g., BAX). In situations
of increased oncogenic activity or mitogenic signals (e.g., activation
of c-Myc or Ras), ARF expression increases. AFR (Alternative Reading
Frame) is a protein resulting from alternative opening of the reading
frame within the p53 gene. ARF protein
binds MDM2, preventing its interaction with p53, leading to p53 stabilization
and induction of apoptosis in response to uncontrolled cell growth. MDM2 is also regulated by Akt kinase, which
phosphorylates MDM2 in response to survival signals, promoting its
transport to the nucleus and potentiating p53 inhibition. In this
way, MDM2 balances between promoting cell survival and responding
to stress.
,
In cancers, MDM2 overexpression
or TP53 mutations disrupt p53 function, driving uncontrolled proliferation
and poor prognosis, especially in treatment-resistant tumors. This dysregulation promotes metastasis, therapy
resistance, and genomic instability. Despite
extensive research, no FDA-approved MDM2 inhibitors exist due to challenges
in balancing efficacy and safety.
,
Targeting
the MDM2-p53 interaction remains a promising strategy to restore p53
function and inhibit tumor growth. The
interplay between MDM2 and p53 in the regulation of apoptosis is summarized
in Scheme
.
The Bcl-2 protein inhibits apoptosis, playing
a key role in carcinogenesis
by promoting cancer cell survival and uncontrolled growth. Its interaction
with p53 regulates apoptosis (Scheme
). In the process of apoptosis,
two main signaling pathways are distinguished: extrinsic and intrinsic
(mitochondrial). The initiators of the intrinsic pathway are mutagenic
factors such as UV radiation, cytotoxic drugs, or oxidative stress,
which leads to DNA damage. DNA damage
activates ATM and ATR, which stabilize p53, triggering the expression
of proapoptotic genes while inhibiting survival-promoting ones. p53
inhibits BCL2, reducing its expression and facilitating the release
of proapoptotic proteins from mitochondria.
,
At the same time, p53 induces the expression of BH3-only proteins
such as NOXA and PUMA. These proteins
neutralize antiapoptotic proteins (e.g., BCL2), blocking their protective
effect, and release proapoptotic proteins BAX and BAK, which become
active. Active BAX and BAK oligomerize in the mitochondrial membrane,
forming pores (MOMP, mitochondrial outer membrane permeabilization),
which leads to the loss of mitochondrial membrane potential. As a result, proapoptotic proteins such as cytochrome
c, Smac/DIABLO, and HtrA2/Omi are released from the mitochondrial
intermembrane space. Cytochrome c initiates apoptosome formation,
while Smac/DIABLO and HtrA2/Omi neutralize inhibitors of apoptosis
(IAPs).
−

Cytochrome c released into the cytoplasm binds to
APAF-1 (Apoptotic Protease Activating Factor 1) and ATP/dATP, which
induces conformational changes of APAF-1 and leads to its oligomerization,
forming the apoptosome – a complex consisting of seven APAF1
molecules. The apoptosome recruits procaspase-9 via CARD domains (Caspase
Recruitment Domain), which leads to its activation through mutual
proteolytic cleavage. Active caspase-9 initiates the caspase cascade,
activating effector procaspases such as caspase-3, caspase-6, and
caspase-7. The caspase cascade leads
to DNA fragmentation and cytoskeletal degradation, forming apoptotic
bodies, which are phagocytosed to prevent inflammation. Overexpression of antiapoptotic Bcl-2 proteins
hinders p53-dependent apoptosis, contributing to cancer cell resistance
to stress-induced apoptosis and cytostatic drugs.
,
The loss of p53 function and high Bcl-2 activity promote cancer
progression and resistance to therapies like chemotherapy and radiotherapy. The mitochondrial pathway of apoptosis involving
BCL2 regulation is depicted in Scheme
.
Apoptosis Inducing Factor Mitochondria Associated
2 (AIFM2), also
known as Ferroptosis Suppressor Protein 1 (FSP1) or as PRG3 (p53-responsive
gene 3), is homologous to AIF, which induces apoptosis independent
of caspases. This mechanism involves
mitochondrial dysfunction and pro-apoptotic factor release, particularly
significant in cancers where classical apoptotic pathways are inhibited
(Scheme
). The exact mechanisms remain unknown, but it
has been found that AIFM2 may perform this function by disrupting
mitochondrial morphology and releasing proapoptotic factors.
−

AIFM2 also acts as an oxidoreductase, protecting cells from ferroptosisa
form of cell death caused by oxidative stress, iron accumulation,
and lipid peroxidation.
,−

AIFM2 reduces ubiquinone (CoQ10) to ubiquinol, an antioxidant stabilizing
cell membranes against oxidative damage. This pathway is independent of glutathione and GPX4, making AIFM2
crucial in alternative ferroptosis protection.
,
Its expression may be regulated by p53, which can inhibit AIFM2
during oxidative stress to promote ferroptosis and tumor cell elimination.
Conversely, under metabolic stress, p53 may support AIFM2 to protect
healthy cells.
,
AIFM2 cooperates with mechanisms
like the ESCRT-III complex, which repairs lipid peroxidation-induced
membrane damage.
,
In cancer cells with inactivated
or mutated p53, AIFM2 may assume a protective role, which promotes
cell survival and may contribute to therapy resistance. Blocking AIFM2,
e.g., with specific inhibitors, is a potential therapeutic strategy
that could increase the sensitivity of cancer cells to ferroptosis
induction. In turn, increasing AIFM2
expression in cancer cells could contribute to the induction of cell
death via apoptosis. Further studies
on AIFM2 may reveal new therapeutic targets for cancer and diseases
linked to oxidative stress. Modulating AIFM2 function could offer
strategies for treating therapy-resistant cancers. The proposed mechanism
of AIFM2-mediated apoptosis and ferroptosis regulation is illustrated
in Scheme
.
RPL4 (Ribosomal Protein L4) is a key component
of the ribosome
that plays an important role in regulating the p53 pathway through
interaction with the MDM2 protein. Studies have shown that RPL4 directly
binds to MDM2, inhibiting its ability to ubiquitinate and degrade
p53, which leads to the stabilization and activation of p53. Additionally, overexpression of RPL4 can lead
to disorders in ribosome biogenesis, resulting in ribosomal stress
and p53 activation. In turn, downregulation of RPL4 induces ribosomal
stress, leading to p53 activation and cell cycle arrest. In the context of cancer, disorders in RPL4
expression can affect p53 function, which is important in the development
and progression of cancer. Studies suggest that understanding the
role of RPL4 in regulating p53 could lead to new therapeutic strategies
for treating cancer. Here, the related
gene of the ribosomal protein RPL41, from the 60S subunit, was taken
as a housekeeping and reference gene followed by RT-qPCR reactions.
Mutations in the p53 protein promote cancer progression. In this
way, the activity of the mutated p53 protein is also changed in relation
to the wild-type p53 protein. Therefore, the p53 protein has become
an ideal therapeutic target. The common
occurrence of mutations in the TP53 gene found in cancers has prompted
research on the p53 protein. Currently, they mainly include restoring
the proper functioning, stabilization or degradation of the faulty
protein.
,
Unfortunately, the development of such a
drug is difficult due to the many possible mutations occurring in
the TP53 gene. Despite this, research on therapy based on the p53
protein has been developing very quickly in recent years.
Phenothiazines
are a class of chemical compounds that have long
been used in medicine, mainly as antipsychotics and antihistamines. Recent studies suggest that some phenothiazines
may have, in addition to other properties, significant anticancer
activity.
−

Their mechanism of action following occurrence may be due to interaction
with p53, which plays a key role in secondary regulation and apoptosis.
Phenothiazines may stimulate apoptosis in cancer cells by activating
p53-dependent pathways. Phenothiazines can affect the interaction
between p53 and its negative regulator MDM2. MDM2 binds to p53, leading
to its degradation. Phenothiazine analogues can block this interaction,
increasing p53 stability and promoting its antitumor functions. Structural modifications to these compounds
may lead to changes in their biological properties, which may be crucial
to increasing their effectiveness as potential anticancer drugs.
−

One strategy for modifying the phenothiazine structure, in
addition
to introducing new substituents to the thiazine nitrogen atom, is
the introduction of various mono- or bicyclic aromatic or heteroaromatic
rings in place of the benzene ring. Our long-standing research on
modifying the phenothiazine system has relied on the modification
of phenothiazines by introducing pyridine, quinoline, and naphthalene
rings in place of the benzene rings (Figure
). As a result of such modifications, we
have obtained a variety of aza- and diazaphenothiazines with the structures
of dipyridothiazines, diquinothiazines, quinobenzothiazines, and naphthoquinothiazines.
Some of these compounds have demonstrated promising immunosuppressive
and anticancer activity against human cancer cell lines derived from
the colon, breast, kidney, lung, ovary, prostate, central nervous
system, melanoma, and leukemia.
,

The tested substances belong to the structural
analogues of phenothiazines,
in which benzene rings were replaced by azine systems (pyridines – BM1, BM2 and quinoline – MJ1, MJ2). Compounds BM1 and BM2 are dipyridothiazines, modified with
pyridine rings, whose correct chemical name is 10H-1,9-diazaphenothiazine­(10H-dipyrido­[3,2-b:2“,3′-e]­[1,4]­thiazine BM1) and 10-propargyl-1,9-diazaphenothiazine­(10-(prop-2-yn-1-yl)-10H-dipyrido­[3,2-b:2”,3′-e]­[1,4]­thiazine BM2) (Figure
). The structure of the compound BM1 was first described in a US patent in 1957. Unfortunately,
the information in the report was very general and did not contain
precise biological aspects. At present, this compound is effectively
obtained in the sulfuration reaction of dipyridylamine using a microwave
reactor. The structure of this molecule was confirmed by NMR, MS,
and X-ray techniques. The BM2 compound was obtained in
the alkylation reaction of 10H-1,9-diazaphenothiazine using propargyl
bromide. Similarly to the BM1 molecule, its structure
was confirmed spectroscopically. Antitumor activities of the BM1 and BM2 compounds were tested. BM1 was a very active substance against the C-32 melanoma line with
the inhibitory concentration (IC) parameter (IC50 = 3.83
μM). On the other hand, the BM2 derivative showed high activity
against the SNB-19 glioma line (IC50 = 3.85 μM) and
C-32 melanoma (IC50 = 3.37 μM). Preliminary TP53
gene expression analysis studies on BM1 and BM2 compounds indicated that these molecules had the ability to disrupt
gene expression in SNB-19, C-32, and MDA-MB-231 cells.

The next two compounds are tetracyclic phenothiazines
in which
one of the benzene rings has been replaced by a quinoline system,
and the benzene ring has a fluorine substituent (Figure
). The MJ1 compound
(6H-9-fluoroquino­[3,2-b]­benzo­[1,4]­thiazine) was obtained
by annulation of 2,2′-dichloro-3,3′-diquinolyl disulfide
with p-fluoroaniline. The MJ2 derivative (9-fluoro-6-propargyl-quino­[3,2-b]­benzo­[1,4]­thiazine)
was obtained by alkynylation of the MJ1 compound using
propargyl bromide. The cytotoxic and
immunomodulatory activity of the MJ2 molecule has been
described in the literature. It also
showed activity against the following cancer lines with the growth
inhibition (GI) parameter: lymphoma L1210 (GI50 = 2.28 μg/mL),
colon: SW948 (GI50 = 44.50 μg/mL), CX-1 (GI50 = 10.83 μg/mL),
and skin cancer A-431 (GI50 = 2.84 μg/mL) Table
.
The compounds for this study were selected due to
the analogy in
the structure of the substituents at the thiazine nitrogen atom, as
well as promising preliminary results for antitumor activity in in vitro studies. Critical to the study of phenothiazines
is their potential interaction with p53, a key regulator of cell cycle
and apoptosis. Strutural changes in phenothiazines can affect the
activation of p53-dependent pathways, such as BCL2 and MDM2 gene expression,
making these compounds promising candidates for the treatment of cancers
with wild-type p53. At the same time,
other modifications may be more effective in cancers with p53 mutations,
opening up new therapeutic possibilities.
,
Phenothiazine derivatives with modifications at the 10-position
of the ring, similarly to compounds BM1 and BM2, were tested for their
ability to generate reactive oxygen species (ROS), which may lead
to the induction of p53-dependent apoptosis. In turn, MJ1 and MJ2 are distinguished by the presence of propargyl
and halogen substituents, which, according to studies, may increase
their affinity for DNA repair proteins and modulate the activity of
protein kinases involved in the p53 pathway. The choice of these four compounds was also dictated by their diverse
lipophilicity and solubility profiles, which allowed for the assessment
of the influence of physicochemical parameters on their biological
activity. Often, the limitation of phenothiazines is their low solubility,
which affects bioavailability and requires the use of higher concentrations,
increasing the risk of adverse effects. This problem highlights the need for further research on improving
the pharmacological properties of phenothiazines, such as changing
lipophilicity or introducing hydrophilic systems, which may influence
their biological activity. The present study aimed to investigate
how these diverse chemical features influence the activation or inhibition
of the p53 pathway in cancer cells. The images presented in Tables
– depict the
results of incubation with the compounds at a concentration of 100
μM, where precipitation was observed. However, for the MTT assay,
appropriate dilutions of the compounds were used, effectively eliminating
precipitation while ensuring accurate determination of IC50 values.
Understanding the role of p53 protein in carcinogenesis
and the
possible influence of phenothiazines on its regulators has been considered
important for the development of medicine. The aim of this work was
to investigate the influence of selected structural analogues of inhibitors
of p53-dependent signaling pathways.

2
Results
The study began with the MTT
test to determine the IC50 parameter values for the tested
phenothiazines, and then microscopic
observations and real-time polymerase chain reaction (RT-PCR) were
performed.
2.1
MTT Colorimetric Test
The cytotoxicity
of selected chemical compounds, BM1, BM2, MJ1, and MJ2, was analyzed using a colorimetric
assay. The HCT116 cell lines with wild-type p53 protein status, HCT116
with mutated p53 protein were used for the experiment. Incubations
of cells with compounds were carried out for two time periods: 24
and 72 h. The IC50 values were determined based on averaged
cell viability data, representing the half-maximal concentration required
to inhibit cell growth. The obtained results are presented in Table
.
Based on the
performed MTT colorimetric tests, IC50 values were calculated
for chemical compounds designated as BM1, BM2, MJ1, and MJ2. The IC50 value
is the concentration of the compound required to inhibit cell viability
and proliferation by 50% compared to the control, which was not exposed
to the compound. Of all the tested compounds, MJ2 showed
the lowest IC50 values in all tested cell lines. Despite
the lowest values, the IC50 remains high. In turn, the BM1 compound had the least favorable IC50 values.
The MTT tests performed show that the tested chemical compounds are
not the best cytostatics for selected cancer lines, because high concentrations
are required to inhibit cell growth. Such doses pose a risk of low
safety of using these compounds against healthy cells. Despite this,
the tested compounds are characterized by cytotoxicity for cancer
cells, and the obtained IC50 values were used for further
experiments.

2.2
Confluency
Cell confluency was evaluated
after 72 h of treatment with the test compounds. Representative images
and quantitative data for HCT116 cells treated with 100 μM of MJ1, MJ2, BM1, and BM2 are presented below.
During the observations, morphological
changes were observed in cancer cells treated with the tested substances,
such as cell shrinkage, cytoplasmic thickening, and cell separation
from the rest, which indicates cell death. The observed changes may
suggest the occurrence of apoptosis, or programmed cell death. At higher concentrations of MJ1 (50 μM, 75 μM, and 100 μM)
and MJ2 (75 μM and 100 μM),
precipitation of the compounds was observed, which may have led to
an overestimation of confluency due to false-positive signal detection.

2.3
Real-Time Polymerase Chain Reaction (RT-PCR)
The key experiment performed was the real-time polymerase chain
reaction. The studies were conducted for the reference gene RPL41
and the genes studied: AIFM2, BCL2, and MDM2. Two HCT116 lines were
used to compare the effect of p53 protein status on the relative increase
in expression.
2.3.1
Experiment 1
RT-PCR was performed
on the HCT116 cancer line with a wild-type p53 protein status (HCT116+/+)
and HCT116 with a mutated p53 protein (HCT116–/−). The
reference RPL41 gene and the p53 gene were selected for experiment
1. Samples incubated for 24 h were selected as a control sample, and
samples after 72-h incubation were selected as the test sample. After
calculating the relative expression level for both cell lines, a high
expression level for the p53 gene was demonstrated in the HCT116 line
with a wild-type p53 protein status (Chart
).
Results are presented as mean ± SD, calculated
from three
experiments and compared to the untreated controls at the 24 h time
point. Statistical significance was calculated using the t-test where p < 0.05 and is indicated by an asterisk (*); bolded
in the table below.

2.3.2
Experiment 2
RT-PCR concerned the
HCT116 cell line with wild-type p53 protein status and HCT116 with
mutated p53 protein. The reference gene RPL41 and the tested genes
were used for the experiment. The studies were performed for the compounds MJ1, MJ2 and BM1, BM2 (administered in IC50 doses), after 24 h of incubation.
2.3.2.1
AIFM2
RT-PCR was performed on
the HCT116 cell line with wild-type p53 protein status and HCT116
with mutated p53 protein. The reference gene RPL41 and the tested
AIFM2 were used for the experiment. The studies were performed for
the compounds MJ1, MJ2 and BM1, BM2 (administered in IC50 doses), after
24 h of incubation (Chart
).
Results are presented as mean ± SD, calculated
from three
experiments and compared to the untreated controls. Statistical significance
was calculated using the t-test where p < 0.05
and is indicated by an asterisk (*); bolded in the table below.
For experiment 2, AIFM2 was used as a test gene, which encodes
an apoptosis-inducing factor associated with canonical activation
of the mitochondrial death pathway. For
the line with mutant p53 protein, all compounds resulted in silencing
of AIFM2 expression. In comparison to the line with wild-type p53
protein, the relative expression level increased.

2.3.2.2
BCL2
Experiment 3 RT-PCR was
performed for the HCT116 cell line with wild-type p53 status and HCT116
with mutated p53. The reference gene RPL41 and BCL2 were used for
experiment 3 (Chart
). BCL2 acts as a regulator of apoptosis for the p53-dependent signaling
pathway.

Results are presented as mean ± SD, calculated
from three
experiments and compared to the untreated controls. Statistical significance
was calculated using the t-test where p < 0.05
and is indicated by an asterisk (*); bolded in the table below.
For the experiment in which the expression level of the BCL2 gene,
which acts as a regulator of apoptosis, was measured, a decrease in
expression was observed for compounds MJ1, MJ2, BM1, and BM2 in the case of the HCT116
line with mutated p53 protein. The highest relative increase in gene
expression was obtained for the BM2 compound of the HCT116
line with wild-type p53 protein status. For the remaining compounds,
the expression level was maintained within the control level. This
means that the HCT116 line with mutated p53 protein has a lower expression
level of BCL2 in contrast to the line with wild-type p53 protein.

2.3.2.3
MDM2
RT-PCR was performed for
the HCT116 cell line with wild-type p53 protein status and with mutated
p53 protein. The reference gene RPL41 and the studied gene MDM2 were
used for the experiment (Chart
).
Results are presented as mean ± SD, calculated
from three
experiments and compared to the untreated controls. Statistical significance
was calculated using the t-test where p < 0.05
and is indicated by an asterisk (*); bolded in the table below.
MDM2 is also known as E3 ubiquitin-protein ligase. For the line with wild-type p53 protein, a clear increase
in the relative expression level is visible after incubation with
the MJ1 compound, and comparable values for the remaining
compounds, compared to the control. For the cancer line with mutated
p53 protein, the expression level is muted for all tested compounds.

3
Discussion
The development of medicine
and pharmacology forces scientists
to create new, safe and effective drugs whose mechanisms of action
are well understood. In order to avoid undesirable consequences of
using drugs and various side effects, many different tests are performed
to check their safety. Cell cultures come to the rescue, which are
relatively cheap, simple and easy to control, in contrast to in vivo
animal or clinical studies. They provide information on the cytotoxicity
and biological activity of drugs. The cytotoxic test, which is the
MTT test, allows for the assessment of the cytotoxicity of selected
chemical compounds, their ability to affect the viability, growth,
and damage of cells. Compounds that have tested and confirmed cytotoxicity
are able to inhibit proliferation and the formation of cell colonies,
stop the cell cycle and DNA replication. Two cell lines were selected for the experiments: HCT116 cell lines
with wild-type p53 protein status and HCT116 with mutated p53. Both
lines produce the p53 protein, and additionally lines with a mutated
protein variant were selected. It was crucial to select these lines
in order to learn about the p53-dependent cellular signaling pathways
and the impact of the tested chemotherapeutics on them.
The
first anticancer activity of phenothiazines was described in
the 1980s. In later years, structures were modified by substituting
various substituents in position 10, which resulted in their anticancer
properties. Modifications of phenothiazines via the pyridine ring
led to the creation of dipyridothiazines. The key aspect for compounds BM1 and BM2, which made them compounds with very
good anticancer properties, is the presence of a dipyridothiazine
scaffold and the arrangement of two azine nitrogens in tricyclic ring
systems. Compound BM2, a derivative of compound BM1 with a substituted propynyl group, is a highly active
compound against glioma lines SNB-19 and melanoma C-32. According
to literature studies, the efficacy of compound BM2 was
confirmed for reducing the expression of the TP53 gene. For the glioblastoma
multiforme cell line, the induction of mitochondrial apoptosis was
observed based on the BAX/BCL2 gene expression ratio. On the melanoma
line, however, this compound affected cell death via the intrinsic
mitochondrial apoptosis pathway.
,,
The MJ1 and MJ2 compounds
are very valuable compounds with high anticancer activity. The modifications
of the MJ2 compound were aimed at reducing cytotoxicity
by substituting the propargyl group in position 6, while maintaining
strong antiproliferative properties.
,,

The first test performed was the colorimetric
MTT test. During
the MTT tests, it was important to maintain the same experimental
conditions for all replicates. The concentrations of the tested compounds,
the time of cell exposure to the drug, the density of cell seeding,
the time of the experiment, sterile conditions in the culture and
other factors played a very key role in obtaining the reliability
and consistency of the results. Another important aspect was the selection
of the appropriate concentration range for the tested compounds. The MTT test is an easy and safe test, characterized
by high repeatability. Based on the colorimetric MTT tests, the IC50 values were calculated for the tested chemical compounds,
marked with the symbols BM1, BM2, MJ1, MJ2. The IC
50
value
means the concentration of the compound that is able to inhibit cell
viability and proliferation by 50% compared to the untreated control.
The best IC50 values for all lines are found for the MJ2 compound. For the HCT116 line with wild-type p53 protein
IC50 = 36.37 μM and IC50 = 57.82 μM
for HCT116 with mutated p53 protein. Despite obtaining the lowest
values, IC50 are still relatively high. The worst activities,
i.e., concentrations with the highest effective values, are possessed
by compound BM1 for the HCT116 line with mutated p53
protein, IC50 = 261.90 μM, for the HCT116 line with
wild-type p53 protein IC50 = 197.25 μM. The MJ1 compound for the HCT116 line with wild-type protein has
IC50 = 56.31 μM, for the HCT116 line with mutated
protein IC50 = 80.78 μM. On the other hand, the BM2 compound for the HCT116 line with wild-type p53 protein
status IC50 = 82.89 μM and for the HCT116 line with
mutated p53 protein IC50 = 87.13. The MTT test conducted
allows us to conclude that for selected cancer lines, the tested chemical
compounds are not the best cytostatic. The concentrations of compounds
that must be administered to inhibit cell growth are very high. The
obtained IC50 results, combined with the morphological
observation of dead cells, may indicate that the apoptotic processes
induced by phenothiazines may be delayed; therefore, the IC50 may be relatively high, indicating the need for longer contact with
the drug to achieve the full cytotoxic effect.
When assessing
the confluence using a JuLI FL Live fluorescence
microscope from NanoEntek, the selected HCT116 cell line showed the
precipitation of the compound for the MJ1 compound at
concentrations of 50 μM, 75 μM, and 100 μM, and
for the MJ2 compound at concentrations of 75 μM
and 100 μM. This could have been caused by the high concentration
of the compounds. The pH of the culture could also have had an adverse
effect. Long-term cell culture (conducted for 24–72 h) has
an acidic pH, which could have affected the precipitation of the compounds
over time, especially at higher concentrations. However, for all tested
compounds, the confluence was significantly lower compared to the
control. Morphological assessment also revealed changes between cells
not treated with the tested compounds and cell lines after the use
of the tested analogues, which confirms the cytostatic effect of phenothiazines
(Tables
–). Despite the high IC50 values obtained,
the morphological changes suggest the induction of cell death already
at lower values (3.15 μM) of all tested compounds. The discrepancy
between the IC50 values obtained in the MTT assay and the
changes observed microscopically may be related to the limitations
of the MTT assay, which measures only metabolic activity, not taking
into account early morphological changes indicative of cell death.
The results suggest that the morphological effects of phenothiazines
on cells appear already at sublethal concentrations, which may be
related to the activation of apoptotic mechanisms or cellular stress
before the overall metabolic activity is significantly reduced.
The key experiment performed was the real-time polymerase chain
reaction. The studies were conducted for the reference gene RPL41
and the genes studied: AIFM2, BCL2, and MDM2. Two HCT116 lines were
used to compare the effect of p53 protein status on the relative increase
in expression. For the purpose of summary, a Table
was created showing the inhibition or lack
of inhibition of expression for selected genes, based on the genes
expression from Charts
–.
AIFM2 plays a key role as a regulator of apoptosis
in the p53-dependent
signaling pathway. This association underscores the importance of
apoptosis as one of the primary mechanisms by which the tumor suppressor
p53 exerts its effects. Importantly, AIFM2 is tightly bound to p53
and its expression is directly regulated by this protein. As a result,
apoptosis-inducing factor 2 plays a significant role in tumor development,
underscoring its importance in cancer biology. For the HCT116 tumor line with the status of wild-type
p53 protein and the tested AIFM2 gene, the relative expression level
for all tested compounds was higher than the control sample. The interaction
of p53 with AIFM2 may be important in this case. For HCT116 lines
with mutated p53 protein, the expression for all compounds was low,
which suggests that the expression was silenced. BCL2 as the next
gene tested belongs to the group of protooncogenes. Mutated p53 protein
has very strong links with BCL2. Strong genetic, chemical and signaling
connections between them are of fundamental importance for oncology.
Interacting proteins play a key role in the process of carcinogenesis
and drug response. BCL2 is mainly responsible for the carcinogenesis
of hematological malignancies. During
the RT-PCR experiment for the HCT116 line with mutated p53 protein
and the MJ1 and MJ2 compounds, an obvious
silencing of the expression level was observed, which means that the
tested compounds inhibited the expression of this gene. The compounds
tested turned out to be effective inhibitors. For the same cancer
line, slightly worse results, but still lower than the control sample,
were obtained by the BM1 and BM2 compounds.
These results confirm the reduction of BCL2 gene expression by selected
compound, which was confirmed in previous studies. For the HCT116 line with wild-type p53 protein, the expression
level was higher for all tested compounds than for the control sample.
This means that the compounds did not inhibit BCL2 expression with
the participation of wild-type p53 protein. The MDM2 gene was chosen
for the last experiment. This is another gene that strongly interacts
with the p53 protein. Unfortunately, regardless of the p53 protein
status, MDM2 can cause various damages and delays in repair. For the
HCT116 cell line with wild-type protein, the relative expression level
for all tested compounds did not decrease. All results are higher
than the control sample. However, for the line with the mutated p53
protein, the expression level decreased for all compounds. For the MJ2 and BM2 compounds, the expression is completely
silenced. This means that these compounds inhibited the expression
of the MDM2 gene, which affects the regulation of the p53 protein.

4
Materials and Methods
4.1
Chemical Synthesis
The tested dipyridothiazines
BM1, BM2 and quinobenzothiazines MJ1 and MJ2 were synthesized
and characteristed according to previously described literature procedures.

4.2
Cell Culture Management
The experiments
utilized well-established HCT116 cell lines, which included two types:
one with functional p53 and the other without p53. The HCT116 cell
line lacking the TP53 gene, created through a biallelic knockout method was kindly provided by Dr. B. Vogelstein. The
HCT116 cell line is a human colon cancer cell line isolated from an
adult male tumor. The HCT116 in vitro cell line is an excellent tool
for studying the mechanisms of tumor growth and metastasis. It is
also useful in assessing the effects of drugs. The model of this line
very well mimics the properties and structure of solid tumor cells.
During the experiments, studies were conducted on the tumor line:
• HCT116 with wild-type p53 protein status (HCT116 p53+/+);
• HCT116 with mutated p53 protein (HCT116 p53–/−).
A vessel with selected cell lines (HCT116 p53 wild type, HCT116
with mutated p53), was taken from the cell bank. Cells were thawed
at room temperature. Using an automatic pipette, the contents of the
vessel were transferred to a 15 mL falcon and suspended in 3 mL of
fresh DMEM culture medium. Then, centrifugation for 3 min (2000 rpm).
After centrifugation was completed, the old medium was removed from
above the cells and 3 mL of fresh medium was added again. The obtained
cell suspension was transferred to a new, appropriately labeled culture
bottle.
The selected cell lines were cultured in plastic culture
bottles
selected appropriately depending on the type of cell line. The bottles
were stored in specially designated incubators with an appropriate
constant temperature of 37 °C and a CO2 concentration
of 5%. The humidity inside the incubator was maintained at about 80%,
which was intended to prevent the cultures from drying out. DMEM culture
medium was used for all studies.

4.3
Confluency
Cell confluency was monitored
using the JuLI FL Live fluorescence microscope (NanoEntek), which
enables real-time imaging and automatic confluency calculation based
on brightfield images. For each experiment, 3 × 105 HCT116 cells were seeded per well in 6-well plates and incubated
under standard conditions (37 °C, 5% CO2). After cell
attachment, the cells were treated with 100 μM of MJ1, MJ2, BM1, or BM2 and incubated for 72 hours.
At the end of the incubation
period, images were acquired using the JuLI FL system and confluency
was automatically calculated using the integrated software.

4.4
MTT Test
The MTT test is classified
as a biochemical test. It is used to assess the cytotoxicity of selected
chemical compounds on cells. This test is based on the ability of
mitochondrial dehydrogenase to convert a tetrazolium salt to a water-insoluble
formazan. The amount of the reduced MTT reagent is directly proportional
to the oxidative activity of mitochondria. Therefore, this test indicates
cell viability (metabolic activity). The product of this reaction
is insoluble in water, which is why organic solvents such as DMSO
or acidic isopropanol are used.
The IC50 (inhibitory
concentration) coefficient is used to express cytotoxic activity.
This is the concentration at which cell viability or proliferation
is inhibited by 50%, in relation to the control sample.
In order
to perform the MTT colorimetric assay, 96-well plates
were first prepared on which selected cell lines were seeded in the
amount of 10 thousand per well. For this purpose, old medium was poured
out from selected culture bottles containing HCT116 lines with wild-type
p53 protein status and HCT 116 with mutated p53 protein. 3 mL of PBS
was added, which was used to rinse the bottom of the bottle and poured
out. Then, 3 mL of trypsin was added to the adherent lines and placed
in incubation for 3–4 min. After the specified time, the bottle
was removed from incubation and the condition of the cells was checked
under a microscope. A double volume of culture medium was added to
neutralize trypsin. The whole was carefully pipetted. Twice 10 μL
of the mixture was taken and applied to the prepared Bürker
chamber from both sides. The Bürker chamber was placed under
a microscope and the sum of cells from the appropriate squares was
counted. After determining the number of cells in the suspension,
it was supplemented with culture medium according to the calculations.
In the next step, 100 μL of culture medium solution with selected
cell lines was added to the prepared 96-well plate. The cells were
incubated for 24 h at 37 °C.
Chemical compounds (BM1, BM2, MJ1, MJ2)
were dissolved in 1 mL of DMSO. After adding
DMSO, the whole was shaken until dissolved for about 10 min. To improve
solubility and ensure complete dissolution, the solutions were subjected
to brief sonication in a water bath sonicator for 10 min at room temperature.
Then new falcons were prepared and volumes of compounds corresponding
to 0.1 mM were added and DMEM medium was supplemented to 5 mL. After
24 h, the medium was removed from all wells of the incubated 96-well
plates. Then, the solutions of chemical compounds (BM1, BM2, MJ1, MJ2) were added
in appropriate amounts. The concentrations of the tested compounds
were in the dilution ranges from 3.15 μM to 100 μM. The
experiment was set up in two time periods: 24 and 72 h at 37 °C.
After the time had elapsed, the entire culture medium was collected
from all plates. 50 μL of MTT reagent (concentration 0.5 mg/mL)
((3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) was
added to each well and incubated for 2–3 h. Then the MTT reagent
was collected and 75 μL of acidic isopropanol was added. The
whole was pipetted and placed on a thermomixer for 10 min. After the
incubation was completed, the absorbance level was measured using
a spectrophotometer at a wavelength of 570 nm using an Epoch plate
reader. IC50 values were calculated based on three independent
biological experiments, each performed in triplicate. Mean IC50 values were determined using linear interpolation, and the
results are presented as mean ± standard deviation (SD).

4.5
RT-PCR
The real-time PCR method is
also known as real-time polymerase chain reaction. The PCR diagnostic
method is one of the most commonly used methods to identify and replicate
a selected nucleic acid fragment.
,
For the polymerase
chain reaction, culture plates were prepared on which HCT 116 cell
lines with wild-type p53 protein status and HCT 116 with mutated p53
protein were incubated. The culture was carried out in two time periods:
24 and 72 h at 37 °C. Control cultures and experimental cultures
were prepared, to which compounds at concentrations equal to the IC50 value were added. After the appropriate incubation time,
the entire medium was collected from the cell cultures into the falcons.
The plates were washed with 1 mL of PBS and transferred to the falcons.
Then, 1 mL of trypsin was added to detach the cells from the bottom
and they were placed for 2–3 min for incubation. Neutralization
was carried out with the contents of the falcon and everything was
placed back into it. The falcon was placed in a centrifuge and spun
for 3 min (2000 rpm). After centrifugation, all supernatant medium
was removed and 400 μL of phenosol was added under a fume hood
using filter tips. The whole was carefully pipetted for cell lysis.
Samples were frozen at −20 °C, where they were stored
until further experiment and RNA isolation.
4.5.1
RNA Isolation
RNA isolation began
by thawing previously collected samples. RNA isolation was performed
according to the instructions included with the A&A Biotechnology
Total RNA isolation kit (Total RNA mini, A&A biotechnology). The
experiment began by incubating the samples for 5 min at 50 °C.
200 μL of chloroform, which was not included in the kit, was
added to the lysate. The samples were gently mixed by inverting the
tubes several times and left for 3 min at room temperature. Then they
were centrifuged for 10 min at 12,000 rpm. In the next step, the upper
fraction was collected in a fume hood and placed in new tubes, and
250 μL of isopropanol was added. The whole was mixed thoroughly
and transferred to the minicolumns included in the kit. The minicolumns
were placed in a centrifuge and centrifuged for 1 min at 12,000 rpm.
After centrifugation, the minicolumns were transferred to new 2 mL
tubes and 700 μL of A1 wash solution, which is included in the
total RNA isolation kit, was added. Then, it was centrifuged again
for 1 min at 12,000 rpm. The minicolumns were removed from the tubes
and the filtrate was poured out. 200 μL of A1 wash solution
was added again and centrifuged for 2 min at 12,000 rpm. The columns
were placed in new clean 1.5 mL tubes with appropriate labeling. 50
μL of sterile water, included in the kit, was added to the bed
at the bottom of the minicolumn. The samples were left for 3 min at
room temperature and then centrifuged for 1 min at 12,000 rpm. In
the last step, the minicolumns were removed and the obtained RNA was
stored at −80 °C until further experiment.

4.5.2
Reverse Transcription
In order
to perform reverse transcription, the RNA concentration obtained during
isolation was measured using a NanoDrop 2000 spectrophotometer. For
this purpose, 1.5 μL of material was taken from the samples
and placed in the device. After measuring the RNA concentration, the
maximum amount of cDNA that could be obtained from the sample with
the lowest concentration was calculated. Then, the amount of sterile
water needed for the experiment was calculated. After completing all
calculations, the reaction solution was prepared according to the
instructions and the assumed number of samples used in the experiment.
The reaction solution contains:
• 5x NG cDNA Buffer
– 4 μL,
• 50 μM Oligo­(dT)­20 –
1 μL,
• NG DART RT Mix – 1 μL,
• Sterile water (RNase-free water).
In the next step,
appropriate amounts of sterile water and RNA
(total 14 μL) were added to the tubes. Then, the reaction mixture
(6 μL) was added, which had been prepared earlier. The whole
was vortexed and centrifuged. In the last step, the tubes were placed
in a thermocycler. According to the manufacturer’s protocol,
they were incubated for 1 h at 50 °C, and then incubated for
another 5 min at 85 °C to terminate the reaction. In this way,
cDNA was obtained. After incubation, the samples were placed in a
freezer (−20 °C) and stored until further analysis.

4.5.3
Real-Time PCR
At the beginning
of the experiment, the samples prepared during reverse transcription
(cDNA) were thawed on ice. Then, based on the concentrations obtained
during reverse transcription, the values needed to perform the RT-PCR
reaction were calculated so that 1 ng of cDNA was available per reaction
(well). The experiment was divided into four parts. Two experiments
are presented as an example (Experiment 1 and 2).
Experiment
1 - conducted on a control culture. Two sets of primers were used
for control RT-PCR: for the p53 gene and the reference RPL41 gene.
Volumes of cDNA, Mastermix, and sterile water for two genes for experiment
1:
cDNA (5,2 μL)
MASTERMIX (60 μL)
STERILE
WATER (30,8 μL)
P53 (12 μL)
RPL41 (12 μL)
Experiment 2 - conducted on culture exposed to the test compounds,
BM1. BM2, MJ1, and MJ2. For this purpose, two sets of primers were
used: for the AIFM2/BCL2/MDM2 gene and the reference gene RPL41. Volumes
of cDNA, Mastermix and sterile water for two genes for experiment
2:
cDNA (4.2 μL)
MASTERMIX (60 μL)
STERILE
WATER (31.8 μL)
AIFM2/BCL2/MDM2 (12 μL)
RPL41
(12 μL)
Each reaction was started by mixing cDNA, sterile
water and Mastermix.
The whole was vortexed and centrifuged. The samples were divided into
2 Eppendorfs, 48 μL each. Each sample was for one pair of primers.
Then the appropriate primers were added in a volume of 12 μL.
After adding the sample, it was vortexed and centrifuged again. In
the next step, an RT-PCR plate with the appropriate number of wells
was prepared. Twenty μL of the mixture was carefully applied
to each well. After the entire application, the RT-PCR plate was carefully
sealed and centrifuged for 3 min at 3000 rpm. After centrifugation,
the plate was placed in a thermal cycler. The experiment was carried
out according to the protocol (Brilliant III Ultra-Fast SYBR Green
QPCR Master Mix, Quick Reference Guide for the Bio-Rad CFX96 Real-Time
PCR Detection System).
Reaction profiles and results for both
the tested and reference
genes were performed using Bio-Rad CFX Maestro 1.1 software from Bio-Rad.
Analysis and calculation of the results obtained during RT–PCR
were performed using Microsoft Excel. For the obtained Cq (Ct) threshold
results, the relative expression level was calculated. The test sample
contained cancer cells incubated with the tested compounds, and the
control sample contained only cells incubated in the medium. The R = 2−ΔΔCt method was used for calculations.
This method allows for the calculation of the relative expression
level (R). It is based on the fluorescence intensity of the selected
gene at the Cq cutoff point.

R – relative level of gene expression,
ΔCq tested – difference in threshold values (Cq) for
the tested gene,
ΔCq reference – difference in
threshold values (Cq)
for the reference gene.
For selected genes used during the real-time
PCR experiment, Table
was prepared, which
contains the primer sequences BCL2, MDM2, AIFM2, and RPL41.

4.6
Statistical Analysis
All biological
experiments were repeated at least three times, with three technical
repetitions each. The data were shown as means ± standard error
(S.E) of three assays. Student’s t-test was applied, and p < 0.05 value was considered as statistically significant.
The statistical significance is indicated by star (*) on the Charts
–, and the changes are compared to the untreated
controls, respectively. For the calculations the MS office version
2010 of Excel was used.

5
Conclusions
The aim of the work was
to investigate the effect of selected structural
analogues on inhibitors of p53 protein-dependent signaling pathways.
Several tests were performed during the experimental studies. The
first study was the MTT colorimetric test. It allowed calculating
the IC50 values for the tested compounds MJ1, MJ2, BM1, BM2. The results
obtained during the test were not satisfactory compared to the literature
values. Despite high IC50 values, morphological changes
indicate induction of cell death already at lower concentrations (3.15
μM). This discrepancy is due to the limitations of the MTT assay,
which does not take into account early morphological changes. The
results suggest that phenothiazines induce apoptotic effects or cellular
stress already at a sublethal stage, before metabolic activity decreases.
The key experiment was the real-time polymerase chain reaction.
These experiments aimed to determine the relative expression level
of selected genes on cell lines containing two different p53 protein
statuses. This allowed determining the effect of the tested compounds
on selected lines and genes. The results confirm the effect of the
p53 protein status on the level of gene expression.
The conclusions
from the conducted experiment indicate a significant
relationship between the status of the p53 protein and the response
of HCT116 cells to phenothiazines. It was observed that in cells with
a functional, wild-type p53 protein, treatment with phenothiazines
led to increased expression of the AIFM2, BCL2, and MDM2 genes, which
are involved in the mechanisms of apoptosis and control of genome
stability. On the other hand, in cells with mutated p53, phenothiazines
caused the opposite effect, reducing the expression of these genes.
These results suggest that the activity of the p53 protein plays a
key role in modulating the cellular response to phenothiazines. In
cells with wild-type p53, the expression of AIFM2, BCL2, and MDM2
increased in response to phenothiazines, which may indicate the activation
of caspase-independent apoptosis mechanisms, in which p53 plays a
regulatory role. In turn, in cells with mutated p53, reduced expression
of these genes after treatment with phenothiazines may indicate a
reduced ability to activate apoptotic pathways, which may contribute
to resistance to treatment.
The reduced response in cells with
mutated p53 may also suggest
that p53-dependent molecular mechanisms are important for the full
activity of phenothiazines in inducing cell death. Furthermore, the
need to use high concentrations of phenothiazines to induce cytotoxic
effects may indicate a potential need for further optimization of
these compounds in the context of cancer therapy, especially in cases
where p53 is mutated or defective.
The conclusions from the
conducted studies indicate that the chemical
structure of the phenothiazines studied plays a key role in their
interaction with the DNA of cancer cells. These phenothiazines exhibit the ability to intercalate into the
DNA structure, which leads to disruption of its integrity and activation
of appropriate DNA repair mechanisms and apoptotic pathways.

Fluoro- and propargyl groups increase
the lipophilicity of these
compounds, which facilitates their penetration into the cells. In
addition, the presence of a propargyl group may affect DNA repair
enzymes and apoptosis mechanisms, contributing to the selectivity
of phenothiazines.
The relationship between p53 status and cellular
response to phenothiazines
suggests that these compounds modify the expression of genes involved
in apoptosis, such as AIFM2, BCL2, and MDM2, depending on the presence
or mutation of p53. In cells with wild-type p53, phenothiazines increased
the expression of these genes, suggesting the activation of caspase-independent
apoptosis mechanisms, whereas in cells with mutant p53, the expression
of these genes was reduced, indicating a reduced response to DNA damage.

In summary, the anticancer and antiproliferative
properties of
the tested compounds were confirmed. These findings indicate that
phenothiazines may be a promising element of future therapeutic strategies,
especially in cancers with mutations in the p53 gene. Understanding
their action in the context of p53 protein status opens new possibilities
in cancer treatment, which may contribute to the development of more
precise and effective anticancer therapies, taking into account the
specific genetic status of cancer cells. Future studies should include
canonical phenothiazines as controls to further delineate structure–activity
relationships and reinforce specificity.

Supplementary Material