BACHELOR’S THESIS
PRECLINICAL EVALUATION OF THE ANTITUMORAL POTENTIAL OF ANTIPSYCHOTIC PHENOTHIAZINES IN BRAIN METASTATIC BREAST CANCER CELLS
Antonio José Serrano Muñoz
Degree in Biochemistry Faculty of Science
Academic Year 2020-21
PRECLINICAL EVALUATION OF THE
ANTITUMORAL POTENTIAL OF ANTIPSYCHOTIC PHENOTHIAZINES IN BRAIN METASTATIC
BREAST CANCER CELLS
Antonio José Serrano Muñoz
Bachelor’s Thesis Faculty of Science
University of the Balearic Islands
Academic Year 2020-21
Key words:
Breast cancer, brain, metastasis, phenothiazines, cell cycle, apoptosis
Thesis Supervisor’s Name Priam de Villalonga Smith Tutor’s Name (if applicable)
The University is hereby authorized to include this project in its institutional repository for its open consultation and online dissemination, for academic and research purposes only.
Author
Supervisor
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Abstract
Breast cancer is one of the most common cancers worldwide, accounting in 2020 for 24.5% of all new cancer cases in women. One of the most aggressive subtypes of breast cancer is triple negative breast cancer, which tends to metastasize to the brain. This type of metastasis implies a poor prognosis for patients, because of the limitations in their management and treatment.
Thus, pharmacological repositioning has been suggested as a possible therapeutic strategy to fight the disease. The aim of this study is to perform a preclinical evaluation of the antitumor potential of two antipsychotic drugs, chlorpromazine and thioridazine, which belong to the phenothiazine family and are commonly used for the treatment of mental disorders such as schizophrenia. To achieve this, a brain metastatic triple negative breast cancer cell line (MDA- MB 231BR) and its parental counterpart (MDA-MB 231) have been used. Our results show a clear antitumoral effect of both drugs since they reduce cell viability and induce the activation of apoptosis. In addition, chlorpromazine induces a cell cycle arrest in G2/M, whereas thioridazine induces a G0/G1 arrest. All in all, the effects of these antipsychotic phenothiazines on triple negative and brain metastatic breast cancer cell lines indicate their possible use as an adjuvant therapy in the management of brain metastatic breast cancer.
Resum
El càncer de mama és un dels càncers més freqüents al món, representant al 2020 el 24,5% del total de nous casos de càncer en dones. Un dels tipus més agressiu de càncer de mama és el triple negatiu, el qual té predilecció per a metastatitzar al cervell, implicant una prognosi molt limitada per als pacients que el pateixen, ja que les característiques de la metàstasi i del propi tipus de càncer en dificulten la teràpia. D’aquesta manera, s’ha proposat el reposicionament farmacològic com una possible estratègia terapèutica per combatre aquest tipus de càncer.
L’objectiu d’aquest estudi és avaluar de forma pre-clínica el potencial antitumoral de dos fàrmacs antipsicòtics, la clorpromazina i la tioridazina, els quals pertanyen a la família de les fenotiazines i s’utilitzen de forma habitual per al tractament de trastorns mentals com l’esquizofrènia. Per a dur-ho a terme, s’han utilitzat una línia cel·lular de càncer de mama triple negatiu (MDA-MB 231) i una línia cel·lular de càncer de mama triple negatiu amb tendència a metastatitzar al cervell (MDA-MB 231BR). Així, els resultats obtinguts han evidenciat el potencial antitumoral de la clorpromazina i la tioridazina sobre aquestes línies cel·lulars ja que redueixen la viabilitat cel·lular i indueixen l’activació de la mort cel·lular programada o apoptosi. A més a més, la clorpromazina indueix un arrest del cicle cel·lular a les fases G2/M, mentre que la tioridazina ho fa a les fases G0/G1. Tot plegat, es demostren els efectes de les fenotiazines antipsicòtiques sobre el càncer de mama triple negatiu i metastàtic al cervell, indicant la seva possible utilització com a teràpia adjuvant en el tractament de la malaltia.
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Index
1. Abbreviation’s list ... 5
2. Introduction ... 6
2.1 Cancer: General concepts ... 6
2.2 Breast cancer ... 6
2.2.1 Types of breast cancer ... 6
2.2.1.1 Triple negative breast cancer (TNBC) ... 8
2.2.2 Breast cancer brain metastasis (BCBM) ... 8
2.2.3 Breast cancer therapies ... 11
2.2.3.1 Triple negative breast cancer therapies ... 12
2.2.3.2 Brain metastatic breast cancer therapies ... 12
2.3 Phenothiazines ... 13
2.3.1 Chlorpromazine ... 14
2.3.2 Thioridazine ... 15
3. Objectives ... 15
4. Materials and methods ... 15
4.1 Cell culture and reactants ... 15
4.2 Cell viability measurement and IC50 determination ... 17
4.3 Cell cycle analysis by flow cytometry... 18
4.4 Apoptosis analysis by flow cytometry ... 19
4.5 Statistical analysis... 20
5. Results ... 20
5.1 Effect of thioridazine and chlorpromazine on cell viability in TNBC and TNBC brain metastatic cell lines ... 20
5.2 Effect of thioridazine and chlorpromazine on cell cycle profile in TNBC and TNBC brain metastatic cell lines ... 22
5.3 Effect of thioridazine and chlorpromazine on apoptosis induction in TNBC and TNBC brain metastatic cell lines ... 27
6. Discussion ... 30
6.1 The antipsychotic phenothiazines chlorpromazine and thioridazine decrease cell viability of MDA- MB 231 and MDA-MB 231BR cell lines ... 30
6.2 The antipsychotic phenothiazines chlorpromazine and thioridazine induce a G2/M and G0/G1 arrest, respectively, in MDA-MB 231 and MDA-MB 231BR cell lines ... 31
6.3 The antipsychotic phenothiazines chlorpromazine and thioridazine induce apoptosis in MDA-MB 231 and MDA-MB 231BR cell lines ... 31
7. Conclusions ... 32
8. Bibliography ... 33
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1. Abbreviation’s list
- APHT: Antipsychotic phenothiazine - AV: Annexin V
- BBB: Blood brain barrier - BC: Breast cancer
- BCBM: Breast cancer brain metastasis - CaM: Calmodulin
- CPZ: Chlorpromazine
- DMEM: Dulbecco’s modified Eagle medium - DR: Dopaminergic receptor
- ECM: Extracellular matrix - ER: Estrogen receptor
- HER2: Human epidermal growth factor receptor 2 - IC50: Inhibitory concentration
- PBS: Phosphate buffered saline - PHT: Phenothiazine
- PI: Propidium iodide - PR: Progesterone receptor - PS: Phosphatidylserine
- TNBC: Triple negative breast cancer - TRD: Thioridazine
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2. Introduction
2.1
Cancer: General concepts
According to the World Health Organization, “cancer is a generic term for a large group of diseases that can affect any part of the body”1. Thus, cancer must be understood not as a simple disease, but as a complex number of diseases which present common characteristics among them.
It is well known that the process of tumorigenesis comprises different steps, each one ruling progressively the transformation of normal cells into malignant ones. The force driving this transformation corresponds to the numerous genomic and epigenomic alterations that cancer cells can acquire. These alterations provide different types of growth advantages to the cells which allow them to proliferate and become, lastly, a tumour2.
Considering the existence of more than 100 distinct types of cancer2, every single type presents unique treats that will define it in a genetic and phenotypic way. However, these different sorts share some of the mechanisms that allow the deregulation of the circuits that control cell proliferation and homeostasis. Weinberg and Hanahan2 reviewed the wide range of cancer cell genotypes and clustered them into “six essential alterations that collectively dictate malignant growth”, the so called Hallmarks of Cancer: “self-sufficiency in growth signals; insensitivity to growth-inhibitory (antigrowth) signals;
evasion of programmed cell death (apoptosis); limitless replicative potential; sustained angiogenesis;
and tissue invasion and metastasis”. Despite these first hallmarks, which contribute to give an essential basis for the comprehension of the cancer’s cell biology, the same authors3 added four new concepts to its initial formulation. On the one hand, the “enabling characteristics” which allow cancer cells to acquire the hallmarks: “genome instability and tumour-promoting inflammation”. On the other hand, the “permissive attributes”: “deregulating cellular energetics and avoiding immune destruction”.
Cancer is nowadays an increasingly global problem. According to the International Agency for Research on Cancer4 (Figure 1), in 2020 the number of new cancer cases worldwide reached the amount of 19,292,789 and they will increase up to 49.7% in the next 20 years. Moreover, not only the incidence of cancer is estimated to increase throughout the years, but associated mortality will also experience an increment of 62.5%, reaching the total sum of 16,180,202 new deaths in 2040.
2.2
Breast cancer
Breast cancer (BC) is one of the most frequent cancers along with colon and lung cancer. Although BC mortality has decreased in Europe and the USA in the last decades due to early detection5,6, it still constitutes the main cause of mortality in women in cancer terms5–7. Moreover, in 2020, BC represented the 24.5% of all new cancer cases in women8.
2.2.1 Types of breast cancer
Despite its common tissue of origin, BC is considered a complex disease with several and different degrees of intra- and intertumoral heterogeneity7,9.
BC can be classified into different types following different criteria. According to the site, meaning if the cancer spreads or not out of the breast, it can be divided into non-invasive or invasive5:
- Non-invasive BC. It corresponds to a cancer that has not spread out of the lobule or ducts where it started. Two examples of this type of BC are ductal and lobular carcinomas in situ,
7 both referring to a development of atypical cells within milk ducts or breast lobules, respectively, that have not extended out of both structures into the breast tissue.
It is important to note that, although non-invasive BC remains at the initial site, cancer cells can develop and progress into an invasive BC type.
- Invasive BC. In this type of BC, malignant cells from the lobules or ducts migrate into proximity of breast tissue. These abnormal cells can also pass through the breast and extend into different parts of the body via systemic or lymphatic circulation. This type of BC is the most common general carcinoma in women and is also known as metastatic BC. Some types of invasive BC are infiltrating lobular carcinoma, infiltrating ductal carcinoma, medullary carcinoma, and tubular carcinoma.
Figure 1
Estimated number of new cancer cases from 2020 to 2040
Note. Estimated number of new cancer cases for both sexes and all ages from years 2020 to 2040.
Adapted from International Agency for Research on Cancer. World Health Organization. Cancer Tomorrow. All Rights Reserved 20204.
Another sort of classification is the one based in histological features. This stratification was based primarily on the expression of three cell receptors: estrogen receptor (ER), progesterone receptor (PR), and ErbB2/neu/HER2 (Human Epidermal Growth Factor Receptor 2)9. Advances in technology have allowed to discover further complexities, in this way leading the appearance of five distinct BC molecular subtypes based on gene expression clustering6,7,9 and on the existence of two epithelial cells types in the human mammary glands, luminal and basal7: luminal A, luminal B, HER2-enriched, basal like, and normal-like. The characteristics of each subtype, including its cell of origin and the expressed receptors, are summarized in Table 1.
8 This molecular BC subtypes differ in prognosis7 and the classification itself has been useful and has led to better outcomes by guiding the selection of targeted treatments such as hormonal therapy and anti- HER2 targeted therapy7,9. In any case, multiple BC subtypes can coexist in the same tumour, and it is also likely that the mixture of molecular subtypes that form a tumour vary along with the development of the tumour itself, inter-converting into other and different subtypes9.
Table 1
Molecular classification of breast cancer
Subtypes ER, PR and HER2 status Cell of origin Luminal A ER+ or PR+ or both, HER2- Luminal epithelial cell Luminal B ER+ or PR+ or both, HER2+ Luminal epithelial cell
HER2-enriched ER-, PR-, HER2+ Basal/myoepithelial
cell/bipotent progenitor Basal like ER-, PR-, HER2± Late luminal progenitor Normal like Tumours that do not fit into
any other categories Luminal epithelial cell
Note. Molecular classification of BC, according to gene expression clustering and the type of mammary glands epithelial cells, luminal and basal. Adapted from Kumar, P. & Aggarwal, R. An overview of triple- negative breast cancer. Archives of Gynecology and Obstetrics 293, 247–269 (2016)7.
2.2.1.1 Triple negative breast cancer (TNBC)
Triple negative breast cancer (TNBC) is defined as the type of BC lacking the expression of ER, PR and HER2. It accounts for the 10-20% of the invasive BCs and englobes more than one molecular BC subtype. In this way, TNBC is considered a phenotype, and its principal molecular components are basal-like tumours, normal-like tumours, and one molecular subtype that has been recently considered, the uncommon claudin-low subtype7.
TNBC consists of a subgroup of BCs with different and heterogeneous clinical presentations, pathology, behaviour, and response to treatment, being aggressive and usually implicating a poor prognosis. The patients affected by TNBC present unfavourable histopathologic characteristics such as larger tumour size and increased lymph node positivity (invasiveness)7.
One of the most important features of TNBC is its higher capacity to metastasize into the brain, one of the most predilect sites of metastasis for BC, as it will be commented below. Furthermore, TNBC implicates a high chance of recurrence between the first and third year after diagnosis and the survival after the first metastatic event is shorter if compared with the other BC subtypes7.
2.2.2 Breast cancer brain metastasis (BCBM)
Cancer metastases consist of cancerous growths that are located at sites of the body which are far removed from the tissue where the primary tumour first emerged. These metastases are constituted by cancerous cells that left the primary tumour and travelled within blood and lymph vessels through
9 the body10. BC, lung cancer, and melanoma are three of the cancer types with a higher rate of brain metastasis, and all of them are associated with a poor outcome11,12.
Among women with metastatic BC, between a 10% and 30% will develop brain metastases12. Breast cancer brain metastasis (BCBM) occur often in younger women, and some of the BC subtypes have a major trend in metastasizing to the brain, being ER negative, HER2-enriched, and TNBC the most important ones13,14. However, there is evidence that supports the idea of an intrinsic molecular subtype switching in BCBM, relative to primary tumours. One example of this switch is the enrichment with HER2, which could be useful for cancer cells in order to favour the progression of the invasion9. Brain metastases have been detected in different parts of the encephalon, including the parietal, occipital, temporal and frontal lobes, and the cerebellum12. In particular for BCBM, the cerebellum seems to be the most favoured location13.
BCBM must be understood as a multistep process, which comprises different sequential stages that led the progression of some primary tumour cells and their invasion of the surrounding breast tissue, their dissemination into systemic circulation via blood vessels, the extravasation of those cells at the brain, and finally the colonization by these cells giving rise to first micrometastases, in order to finally become macroscopic brain metastases of BC12,15(Figure 2).
As mentioned above, the metastases begin with the invasion of cancerous cells into the surrounding tissue, a process that needs an alteration of the cell-to-cell adhesion and the adhesion to the extracellular matrix (ECM). Changes in the expression of adhesion molecules, such as the downregulation of E-cadherin and the expression of N-cadherin, play a major role in this first invasion step. These changes take place as part of an essential event called epithelial to mesenchymal transition (EMT), when cancer cells with an epithelial phenotype acquire a mesenchymal-like shape, allowing them to escape the primary tumour and invade other parts of the body, interacting with stromal cells15. The principal stromal cells within metastatic BC are fibroblasts, and they are usually referred as carcinoma associated fibroblasts (CAFs)15. During this first step, malignant cells also develop the capacity to degrade the ECM, thus facilitating its path. This degradation is possible thanks to the expression of matrix metalloproteinases (MMPs)14,15.
The blood brain barrier (BBB) consist of a highly selective filter that accomplishes the mission of protecting the brain from potentially harmful agents, such as toxic hydrophilic molecules, an excessive inflammation event due to infiltration of periphery immune cells, or even metastatic cells attempting to access the brain parenchyma11. The sealing of the BBB is ensured by tight junctions between endothelial cells of the brain capillaries. Tumour cells afford the passage through the BBB imitating the mechanisms used by immune cells in their endothelial transmigration process14. An example of this mechanisms is the expression of cathepsin S, a protease which is usually expressed by leukocytes, mediating the transmigration of BC cells through the BBB via degradation of tight junctions11,14. Thus, the extravasation event is based on the initial adherence to the endothelium, the establishment of intracellular contacts, transendothelial migration, adhesion to the subendothelial matrix, and finally, the alteration of the surrounding host tissue (brain). This crucial step is favoured by a reciprocal interaction between cancer cells and stromal cells, and it has been recently noticed that the endothelial cells actively influence the extravasation by expressing different kinds of integrins, selectins and chemokines14.
10 Figure 2
Breast cancer brain metastasis process
Note. Schematic representation of the different steps that take place in BCBM. In the breast, the primary tumour acquires the capacity to invade the surrounding tissue, initiating the metastatic cascade that finalizes with the tumoral cells accessing through the BBB into the brain, forming a micrometastasis that will develop into a macrometastasis. In the latter process, interaction with resident astrocytes is essential. Created with BioRender.com.
11 When BC cells finally arrive in the brain, they must face a complex microenvironment that is quite different from the niche of the primary tumour. This microenvironment entails the most important source of selective pressure on cancer cells, contributing to define which BC cell clones will seed into the brain and lastly give rise to a macrometastasis, or to the contrary, perish and remain unable to regrow the tumour. The brain parenchyma is formed by different cell types such as astrocytes11, which contribute to tissue homeostasis and help to sustain the BBB12,14, microglia, oligodendrocytes, and neurons, and some of them maintain a complex and vast connectome of electrical signs. According to this, breast metastatic cancer cells must interact between these cells to proliferate and be capable of forming a new solid tumour11,12,14,15. Thus, metastatic cells adapt to this microenvironment by adopting
“brain-like” properties11, and interact with astrocytes enabling a cross-linked and heterologous communication. An example of this communication is the one in which tumour cells utilize astrocyte gap junctions to transfer cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) to astrocytes, promoting in them the synthesis of inflammatory cytokines such as interferon alpha (INFα) and tumoral necrosis factor (TNF) that, in turn, will activate in cancer cells some signal transduction pathways like JAK-STAT and NF-κB which in last term will promote tumour growth and chemoresistance12,14. This kind of interactions promote a “switch from a metastasis-hostile brain parenchymal microenvironment to a cancer-promoting one”11.
2.2.3 Breast cancer therapies
Early BC that remains at the same site where it started, without evidence of detectable distant metastasis, is a potentially curable disease. Diagnosis of BC takes place by clinical examination through mammography and breast ultrasound, even confirming the presence of the tumour via core biopsy.
Once cancer is diagnosed, various therapy and treatments concepts need to be decided6. Different strategies of disease management are used for patients with BC, such as surgery, chemotherapy, radiation therapy, hormonal therapy and targeted therapy5.
Although the first initial logical request from the patient would be the surgery of the tumour or even of the breast, currently, breast conservation is technically affordable in many clinic situations that, a few years ago, could not have been faced with any other alternative than primary mastectomy6. In this way, other medical approaches like radiation therapy are useful for reducing the clinically necessity of mastectomies. Moreover, this radiation therapy can be maintained in some cases after the surgery of the tumour to kill the possible remaining malignant cells of the area5.
Some chemotherapeutical agents approved for BC management are Docexatel, Paclitaxel, and Platinum agents such as carboplatin or cisplatin. In any case, the diverse side effects of this kind of therapy are the main reason to find some alternative methods5.
Hormonal therapy such as anti-estrogen therapy is feasible in those BCs that are affected by hormones, thus implicating the expression of hormonal receptors like ER. There are different hormonal therapeutic agents used in this type of treatment: tamoxifen, raloxifene, toremifene, etc. Tamoxifen blocks in a competitive way the union of the hormone estrogen to its receptor (ER), thus inhibiting cancer cells from developing.
Targeted therapies consist of medicaments prescribed to only manage some types of BC, because its molecular target it is only expressed in those subtypes. The principal and more known targeted treatment is the administration of the monoclonal antibody Trastuzumab, commercialized by the name of Herceptine5. This antibody binds specifically to the HER2 receptor and blocks its tumorigenic action in different ways, such as inhibiting the scission or dimerization of the receptor and the consequent signal transduction, promoting the recognition of immune system of the malignant cell, or even inducing the degradation of the receptor via endocytosis16(Figure 3).
12 2.2.3.1 Triple negative breast cancer therapies
TNBC locoregional treatment is similar to the one performed in other invasive BC types, counting with the excision of the tumour mass by mastectomy or by conservation-breast surgery followed by radiation therapy7.
It has to be kept in mind, that TNBC lacks the expression of ER, PR and HER2, thus since these targets are missing, neither hormonal therapy or targeted therapy like Tamoxifen or Trastuzumab, respectively, are available for the management of this kind of molecular BC subtype, leading conventional chemotherapy to be the only systemic treatment which improves the outcome of the patients6,7.
2.2.3.2 Brain metastatic breast cancer therapies
Unlike non-invasive BC, distant metastases like brain metastases are difficult to treat and their management is usually addressed to palliate the devastating derived complications and enhance the life quality and the survival rate of the patients5,6,17. These treatments include radiotherapy, surgery, and steroids. Despite this, the role of molecular drugs in brain metastasis therapy is complicated due to the presence of the BBB, which restricts the access to the brain of many compounds17, decreasing in this way the intracranial response rate of those treatments11.
Figure 3
Potential mechanisms of action of Trastuzumab
Note. Most well-documented potential mechanisms of action of the monoclonal antibody Trastuzumab in the treatment of HER2 positive BC. (B) Molecular structure of the antibody Trastuzumab. (C) Reducing of HER2 extracellular domain shedding by the union of Trastuzumab, blocking the signal transduction activation by the cleaved phosphorylated product p95. (D) Inhibition of the homo- and heterodimerization of the receptor by the union of Trastuzumab. (E) Recruitment of Fc-competent immune effectors cells and other components of the antibody-dependent cell-mediated cytotoxicity by Trastuzumab Fc region. (F) Receptor down-regulation by endocytosis via Trastuzumab union. Adapted from Hudis, C. A. Trastuzumab — mechanism of action and use in clinical practice. New England Journal of Medicine 357, 39–51 (2007).16.
13 Systemic therapy is the first treatment that is usually selected in BCBM6. However, in the case of TNBC brain metastasis, its management is preferentially done with radiation therapy rather than using systemic therapy. Patients with this kind of brain metastasis often show different symptoms like nausea, vomiting or headache, among others, so they are treated with corticosteroids to reduce them during radiation treatment12. Furthermore, and as commented above, TNBC account with a crucial drawback respect to other subtypes as it cannot be managed with hormonal or anti-HER2 therapy, thus hindering brain metastases of this sort of BC12.
In short, although advances in therapies for BC metastasis have improved the survival of patients among last years, metastatic BC still constitute an incurable disease15, and as specific treatments for BCBM have not been developed yet, the prognosis of the disease is nowadays poor12. It seems important to find new treatments that allow the management of both primary tumour and its brain metastasis11, because in many patients systemic response and brain response to the same experimental treatment often do not correlate14. Furthermore, the development of new therapies addressed to prevent and treat TNBC metastases are also on the point of view, being the method of drug repurposing a potential candidate for finding novel drug therapies. This method, which consist in testing the antitumoral potential of drugs that are already on the market for the treatment of other completely different diseases, has been shown to be time-saving and cost-effective18,19.
2.3
Phenothiazines
Phenothiazines (PHTs) are a family of drugs with a common chemical structure that contains a phenothiazine ring, an alkyl connector attached to the N-10, and a terminal amine group. Its molecular structure and high lipophilicity, conferred by the phenothiazine ring, are fit to easily cross the BBB, and exhibit a high affinity to lipid bilayers of the neuron’s plasmatic membrane. Moreover, depending on the structure of substituents in the side chain, PHTs compounds can be classified into three groups, each with different neuroleptic actions: piperazine PHTs, piperidine PHTs, and aliphatic PHTs (Figure 4A)20.
PHTs are used as antipsychotic drugs due to its ability to bind to various receptors in the central nervous system (CNS), in particular blocking dopaminergic receptors (DRs), which consist in the strongest union, but also α-adrenergic, serotonin, muscarinic, γ-aminobutyric acid (GABA)-ergic and histamine receptors. This higher affinity for DRs is due to its three-dimensional structure resemblance with dopamine20, affording the establishment of similar molecular interactions within the receptor (Figure 4B). The family of DRs contains a total of five receptors, named from DRD1 to DRD5, and consist of G-protein-coupled receptors (GPCRs) which mediate the response to the catecholamine dopamine.
In turn, DRs are classified into two groups, the D-1 like receptors (DRD1 and DRD5), and the D-2 like receptors (DRD2, DRD3 and DRD4), and both groups mediate different cellular responses to the union of dopamine. On the one hand, D-1 like receptors are coupled to Gαs proteins and promote the synthesis of cAMP, while on the other hand, D-2 like receptors are coupled to Gαi/o proteins and inhibit the production of cyclic adenosine monophosphate (cAMP), thus having the two types of receptors opposite molecular effects when activated21,22. In particular, DRD2 activity in excess or default is thought to be the cause of different CNSs diseases such as schizophrenia and Parkinson’s disease22.
Despite its evident neurologic effect, PHTs also exhibit a vast range of other biological effects, such as an antitumorigenic effect. It has been described their ability to inhibit calmodulin (CaM), protein kinase C (PKC), and decrease cell proliferation, among others. The substituents (X) linked to the position C-2 of the tricyclic phenothiazine ring and the length of the alkyl connector (Figure 4A) determine the activity and the effect of PHTs against cancer cells20. In this way, there are recent evidences that
14 antipsychotics targeting the DRD2 can inhibit the proliferation and growth of many cancer types, such as leukaemia, glioblastoma, and lung, colorectal and BC cell lines22.
In the present study, we focused our attention on two different antipsychotic phenothiazines (APHTs):
chlorpromazine and thioridazine.
Figure 4
General chemical structure of phenothiazines and its similarity with dopamine
Note. (A) General chemical structure of PHTs family drugs and its classification according to the chemical substituents of N-10. (B) Superposition of PHTs and dopamine chemical structures. Adapted from Jaszczyszyn, A. et al. Chemical structure of phenothiazines and their biological activity.
Pharmacological Reports 64, 16–23 (2012).20. 2.3.1 Chlorpromazine
Chlorpromazine (CPZ) (Figure 5B) is a PHT that belongs to the first generation of antipsychotic drugs23, known for being the first antipsychotic drug used for the management of schizophrenia and bipolar disorder19.
One special characteristic of CPZ is its ability to concentrate within the brain when is administrated. It has been described that CPZ’s brain concentration is four to five times higher than its plasmatic concentration24–26. Thus, the brain CPZ: plasma CPZ ratio is equivalent or even greater than 4:125. This fact turns CPZ into an attractive neuroleptic drug because its administration at lower doses implies a greater effect into the brain due to its pharmacological distribution properties.
CPZ binds to the DR with an antagonistic effect, but it can also bind to other kind of molecules such as CaM, different channel proteins, DNA topoisomerase, oncogenic protein K-Ras, etc. This wide capacity to bind different cellular targets allows CPZ to exhibit different antitumoral effects. For example, it has been demonstrated that CPZ inhibits the proliferation of cancer cells by inhibition of the mitotic kinesin KSP/Eg5, resulting in mitotic arrest, and increases the expression of p21Waf1/Cip1 via the activation of the tumor suppressor Egr-1 or through the inhibition of the PI3K/Akt signalling pathway23. It has also been shown that CPZ is capable of inducing autophagy and apoptosis in brain tumour cells and enhances the efficiency of the treatment of ER negative BC cells. Moreover, recent studies have elucidated that CPZ inhibits BC stem cells proliferation via inducing the signal transduction Hippo pathway19.
A. B.
15 2.3.1 Thioridazine
Thioridazine (TRD) (Figure 5A) is another APHT drug that, similar to CPZ, is used for the treatment of schizophrenia18.
The antitumor potential of TRD has also been reported, being administrated for the treatment of tumour-associated sweating and depression, but also as an effective cancer therapy. In glioblastoma, TRD inhibits the proliferation of glioma stem cells and induces autophagy, whereas in melanoma, it retards tumour growth and decreases tumour-associated vasculature. Moreover, similar effects have also been reported in prostate and gastric cancer, as well as in lymphoma18.
Referring to BC, evidence also supports the positive effect of TRD since it targets the self-renewal of basal-like TNBC18. TRD has also shown its capacity to inhibit the proliferation of some BC cell lines and to induce cell cycle arrest21, and recent studies have demonstrated that this cell cycle arrest in TNBC depends on the union of TRD to its target DRD2, via activation of STAT322.
Figure 5
Chemical structure of thioridazine and chlorpromazine
Note. (A) Chemical structure of TRD. (B) Chemical structure of CPZ.
3. Objectives
The main objective of this study is to determine and evaluate the antitumor potential of different APHTs (CPZ and TRD) in brain metastatic BC cells. Thus, the following procedures will be performed:
1. Establish the antitumor effect of the APHTs CPZ and TRD on the cell viability of two BC cell lines: MDA-MB 231 and a brain-seeking line MDA-MB 231BR.
2. Determine the inhibitory concentration (IC50) of those drugs.
3. Analyse via flow cytometry the effect of the tested PHTs on cell cycle progression and on the induction of apoptosis in the indicated breast cancer cell lines.
4. Materials and methods
4.1
Cell culture and reactants
In this study, two BC cell lines (Figure 6B and 6C) were used to evaluate the antitumor potential of CPZ and TRD:
A. B.
16 - MDA-MB 231. This human TNBC cell line is derived from a pleural effusion. The cells are adherent and present an epithelial morphology. It is also characterized by the expression of EGFR (Epidermal Growth Factor Receptor), TGF-α (Transforming Growth Factor-α) and the oncogene Wnt-7B27.
- MDA-MB 231 Brain (BR). This cell line consists of a MDA-MB 231 subclone that selectively spread to the brain at a histological level, that is, it is brain-seeking. Its establishment was based on the intracardiac injection (left ventricle of the heart) of MDA-MB 231 cells in mice, the subsequent isolation of those cells that had metastasized into the brain, their culture, and their reinoculation. This procedure was repeated six times, and at the end of it, the MDA-MB 231BR clone was established28. The MDA-MB 231BR cells used in this study were a gift from the Patricia S. Steeg Laboratory (Centre for Cancer Research, National Cancer Institute, Bethesda, Maryland), where the previously described brain-seeking cell line was subjected to a seventh intracardiac injection.
To check whether the drugs used in this study (CPZ and TRD) were correctly prepared, an additional cell line, LN229, was treated and processed along with the others (Figure 6A). This cell line derives from a human glioblastoma located at the right frontal parieto-occipital cortex. The cells are adherent and present an epithelial morphology. Moreover, they have the tumoral suppressor p53 mutated, as well as other deletions in tumoral suppressors’ genes such as p16 and pARF29. Previous results from the Cancer Cell Biology Laboratory (Institut Universitari d’Investigació en Ciències de la Salut -IUNICS-, Universitat de les Illes Balears -UIB-, Palma, Illes Balears, Spain) indicate that the cell line is sensitive to the antitumor effects induced by both PHTs, allowing the utilization of these cells as an internal control (unpublished data).
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Figure 6
Cell morphology of LN229, MDA-MB 231 and MDA-MB 231BR cell lines in monolayer
Note. Inverted microscope images of (A) LN229 glioblastoma cell line30, (B) MDA-MB 231 BC cell line, and (C) MDA-MB 231BR metastatic BC cell line. All cell lines were grown in monolayer, as seen on the images.
All cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10%
inactivated foetal bovine serum (FBSi) and 1% penicillin/streptomycin. As commented above, the three cell lines are adherent and grow in monolayer. Thus, periodic passages were performed using trypsin to detach the cells and phosphate buffered saline (PBS) to previously wash them.
A. B. C.
200 µm 200 µm
200 µm
17 During the whole experimental period, all three cell lines were grown in a humified incubator at 37°C with 5% CO2 atmosphere. Moreover, cell lines were treated (during two weeks) with a concentration of 12.5 µg/ml PlasmocinTM after the detection of Mycoplasma contamination31. Once this treatment was completed, the cells were disinfected, and the presence of Mycoplasma was periodically checked.
The two APHTs used in this study (CPZ and TRD) were obtained from Sigma-Aldrich®.
4.2
Cell viability measurement and IC
50determination
To determinate the IC50 of CPZ and TRD on cell lines, cell viability assays were performed.
For this purpose, 2.5 × 103 cells/well were seeded in 96-well plates. After that, treatment with each PHT was performed, according to the following dosage for each cell line and for both drugs: 5 µM, 6 µM, 7.5 µM, 10 µM, 12 µM, 15 µM, 20 µM. During this first part of the experimental procedure, the previous doses were adjusted. Thus, the final concentrations assayed were:
- CPZ: 2.5 µM, 5 µM, 6 µM, 7.5 µM, 10 µM, 12 M, 15 µM.
- TRD: 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, 10 µM.
Each dose was assayed per triplicate. Moreover, for each cell line and drug, three control wells were prepared. The disposition of each 96-well plate is indicated in Figure 7. According to this, for each cell viability experiment two 96-well plates (three cell lines in total) were needed.
Figure 7
96-well plate disposition in cell viability assays
Note. 96-well plate organization for cell viability assays. The control wells were filled only with DMEM.
Created with BioRender.com.
After 48h of treatment, the number of viable cells for each condition was determined via ATP quantification, since its concentration is directly proportional to the presence of metabolically active cells. For this determination, the Cell Titer-Glo® luminescent assay kit (Promega)32 was used. This kit causes the lysis of viable cells, allowing the release of intracellular ATP to the medium, which is subsequently quantified due to luciferase reaction. For the detection of luminescence, a multi-step well Synergy Mix scanning spectrophotometer (Biotek) was used.
18 To determine the IC50, data obtained from cell viability experiments were subjected to curve fitting analysis through non-linear regression using the GraphPad Prism software.
4.3
Cell cycle analysis by flow cytometry
To evaluate the possible effect of both PHTs on cell cycle, flow cytometry cell cycle analysis was performed.
This technique allows the evaluation of cell cycle profile after 24h or 48h PHT treatment. As seen in Figure 8, cellular DNA content varied along cell cycle progression, and this variation can be used to identify the cell cycle phase distribution. When stained, the DNA of cells binds the dye in a stoichiometrically way. Consequently, flow cytometric analysis of cell count in function of dye emitted fluorescence is used to create a histogram. In this way, flow cytometry can be used to estimate the percentage of cells at different cell cycle phases (G0/G1, S or G2/M). Moreover, dead cells can also be quantified, representing the Sub-G0/G1 fraction (the DNA content is lower than the one in diploid cells, due to formation of cell fragments)33.
The DNA dye used in this study was propidium iodide (PI). PI is a membrane non permeant fluorescent dye which is capable of binding to double stranded DNA by intercalating between base pairs. PI is unable to enter viable cells. Thus, only apoptotic cells can be dyed because of membrane alteration and permeability increment. Despite this, to stain the DNA in viable cells, an ethanol fixation was previously performed34.
Figure 8
Cell cycle flow cytometry analysis
Note. (Left) Schematic representation of cell cycle’s phases. Cells can enter cell cycle to duplicate their amount of DNA (from 2n diploid to 4n tetraploid) and then divide by mitosis. (Right) Illustration of a standard cell cycle histogram obtained by flow cytometry. Each cell cycle phase is related to one histogram section, as indicated in the figure. Created with BioRender.com.
To perform the analysis, 1.5·104 cells were seeded in p60 plates. In this experiment, the control LN229 cells were not used anymore, because the sensitivity of both BC cells to PHTs had already been
19 established in cell viability assays. Thus, in this and further experiments only MDA-MB 231 cells (brain and wildtype) were used.
Treatment with PHTs was performed for 24h and 48h. Moreover, one negative control per cell line and time point was also prepared.
PHTs doses used in this experiment were:
- For MDA-MB 231:
o CPZ: 3.75 µM and 5 µM.
o TRD: 5 µM and 7,5 µM.
- For MDA-MB 231BR:
o CPZ: 7,5 µM and 10 µM.
o TRD: 5 µM and 7,5 µM.
To fix the cells after treatment, the media from each plate was collected to keep possible dead cells that had detached from the plate surface. Cells that remained attached were trypsinized and added to the previous ones. At this point, centrifugation of cells and subsequent washing with PBS were performed. After that, another centrifugation was performed, and the resulting pellet was resuspended in PBS and fixed with ice-cold ethanol at a final concentration of 70%. The fixed cells were stored over weekend at 4°C.
Before staining the cells, two washes with PBS were performed. The resulting pellet of cells was stained using a PI staining solution. For each millilitre, the staining solution contained: 50 µl PI (1 mg/ml), 10 µl RNase (5mg/ml) and 940 µl PBS. Finally, cells were analysed through flow cytometry.
4.4
Apoptosis analysis by flow cytometry
To identify possible cells that due to PHT treatment had undergone apoptosis, flow cytometry apoptosis analysis was performed.
Plasma membranes of normal cells account with an asymmetrical distribution of phospholipids. Thus, lipid bilayer components are usually orientated to one side of the membrane in an exclusive way. This is the case of phosphatidylserine (PS), which faces the intracellular space. When cells activate the programmed cell death program or apoptosis, this membrane asymmetry is lost, and phospholipids like PS change their orientation, facing the extracellular space. Moreover, the membrane integrity is also affected in late stages of apoptosis, losing its impermeability to certain compounds, such as PI, which as has been indicated in the previous section, is unable to enter intact cells35.
The detection of apoptotic cells was performed using the Dead Cell Apoptosis Kit with Annexin V Alexa FluorTM 488 and Propidium Iodide (PI), which was obtained from Termo Fisher®. This kit contains the protein annexin V (AV) that specifically binds to PS when exposed to the extracellular surface of plasma membrane. The protein is conjugated to fluorescein isothiocyanate (FITC), allowing the detection via flow cytometry of apoptotic cells. Furthermore, the kit also contains PI, which binds to dead cells’ DNA, allowing the detection of late-stage apoptotic cells. Altogether, the kit allows the detection of early- stage apoptotic cells (AV positive, PI negative) and late-stage apoptotic cells (AV positive, PI positive).
Viable cells will be negative for both fluorophores36.
To perform flow cytometry apoptosis analysis, 1.5·104 cells were seeded in p60 plates. The CPZ and TRD concentrations used in this assay were:
20 - For MDA-MB 231: CPZ 7.5 µM, 10 µM and 12.5 µM; TRD 7.5 µM, 10 µM, and 12.5 µM.
- For MDA-MB 231BR: CPZ 10 µM, 15 µM and 20 µM; TRD 7.5 µM, 10 µM, and 12.5 µM.
To collect the cells, exact procedure as the one for Cell cycle analysis by flow cytometry was performed.
Once the final pellet was obtained after the last PBS wash, it was resuspended with 100 µl Alexa Binding Buffer (ABB), and 5 µl Alexa Fluor 488 annexin V and 1 µl PI solution were added. After a 15- minute dark incubation, 400 µl of ABB were added and the samples were read by flow cytometry at an excitation of 488 nm (FITC channel).
4.5
Statistical analysis
All data in this study are given as means with their respective standard deviation (mean ± SD).
Statistical significance was assessed via one-way ANOVA, and differences between conditions (control and treatments) were assessed by post hoc analysis (Student’s t-test) using the program GraphPad Prism. Statistically significant differences are indicated by p-value: *** p<0.001, **p<0.01 and *p<0.05.
5. Results
5.1
Effect of thioridazine and chlorpromazine on cell viability in TNBC and TNBC brain metastatic cell lines
In order to evaluate the possible antitumor effects of CPZ and TRD in BC and BC brain metastatic cell lines, cell viability assays were performed. After that, dose-response curves of each drug and cell line were represented. Each curve shows cell viability percentage values (taking control values as 100%) in function of the logarithm to base 10 of drug concentration (in M). The resulting curves are shown in Figure 9.
As represented in Figure 9A, the effect of CPZ in MDA-MB 231 and MDA-MB 231BR cell lines differs.
On the one hand, the viability profile of MDA-MB 231 cells shows a greater effect of CPZ at lower doses, being the curve displaced to the left respect the two others. Moreover, the decrease in cell viability is more marked in MDA-MB 231 cells than the one observed for LN229 and MDA-MB 231BR cell lines.
On the other hand, the effect of CPZ in MDA-MB 231BR cells is lower when compared to normal MDA- MB 231 cells. Thus, a higher dose is required to initiate the reduction of cell viability, being the curve displaced to the right. In addition, this reduction is less pronounced at initial dosage.
Referring to TRD (Figure 9B), and contrary to CPZ, all viability cell profiles are quite similar, thus meaning a similar effect of TRD on the three cell lines at same dosage. In this way, MDA-MB 231 and 231BR cells show a similar response to TRD treatment at 48h. Some differences can be observed within -5.5 and -5.2 concentrations (3 and 6 µM, respectively), having TRD a higher effect on MDA-MB 231 cells respect to the metastatic ones (curve slightly displaced to the left). However, at a concentration of 7 µM (-5.1 at the graphic) the effect of TRD on both BC cell lines’ viability is equivalent.
Furthermore, it is important to note that in general terms, TRD presents a higher antiproliferative effect on all cell lines respect to CPZ, thus achieving a reduction of cell viability in practically 100% at a concentration of 10 µM (-5 at the graphic), while at the same concentration some cells still remain viable for CPZ treatment.
21 Figure 9
Cell viability dose-response curves of LN229, MDA-MB 231 and MDA-MB 231BR cell lines after the treatment with CPZ and TRD
Note. (A) Dose-response curves result of CPZ treatment during 48h in LN229, MDA-MB 231 and MDA- MB 231BR cell lines. Each value corresponds to the mean of cell viability in function of the logarithm to base 10 of CPZ concentration (2.5 µM, 5 µM, 6 µM, 7.5 µM, 10 µM, 12 µM and 15 µM). SD for each value is also represented. Each curve is the result of 3 independent assays. Statistical significance of the differences between treatments and control is represented: *p<0.05, **p<0.01, ***p<0.001. (B) Dose-response curves result of TRD treatment during 48h in LN229, MDA-MB 231 and MDA-MB 231BR cell lines. Each value corresponds to the mean of cell viability in function of the logarithm to base 10 of TRD concentration (3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM and 10 µM). SD for each value is also represented. Each curve is the result of 3 independent assays. Statistical significance of the differences between treatments and control is represented: *p<0.05, **p<0.01, ***p<0.001.
A.
B.
CPZ
Comparation LN229 MDA- MB 231
MDA-MB 231BR
Control vs. 2.5 µM ** *** ns
Control vs. 5 µM *** *** **
Control vs. 6 µM *** *** ***
Control vs. 7.5 µM *** *** ***
Control vs. 10 µM *** *** ***
Control vs. 12 µM *** *** ***
Control vs. 15 µM *** *** ***
Log (M) Chlorpromazine
% Cell Viability
-6.0 -5.5 -5.0 -4.5
0 20 40 60 80 100 120
LN229
MDA-MB 231BR MDA-MB 231
TRD
Comparation LN229 MDA- MB 231
MDA-MB 231BR
Control vs. 3 µM * * ns
Control vs. 4 µM * * ns
Control vs. 5 µM *** *** **
Control vs. 6 µM ** ** ***
Control vs. 7 µM *** *** ***
Control vs. 8 µM *** *** ***
Control vs. 10 µM *** *** ***
-6.0 -5.5 -5.0 -4.5
0 20 40 60 80 100 120
Log (M) Thioridazine
% Cell Viability
LN229
MDA-MB 231BR MDA-MB 231
22 Besides dose-response curves, IC50 was calculated for both drugs on each cell line. The term IC50 refers to the concentration of a drug that is required to produce a 50% of inhibition37. In this study, the IC50
refers to the concentration of each drug required to reduce cell population in a 50%. Results from the calculation of TRD and CPZ’s IC50 are shown in Table 3.
Table 3
IC50 values of TRD and CPZ in LN229, MDA-MB 231 and MDA-MB 231BR cell lines
IC50 (µM) LN229 MDA-MB 231 MDA-MB 231BR
Thioridazine (TRD) 5.19 ± 0.23 5.06 ± 0.29 5.94 ± 0.27 Chlorpromazine (CPZ) 5.91 ± 0.47 4.34 ± 0.61 9.15 ± 0.92 Note. Mean and SD is represented for each drug and cell line. IC50 values are the result of 3 independent experiments.
These results support the observations based on dose-response curves. Calculated IC50 values show a more robust effect of TRD on MDA-MB 231BR cell lines when compared to CPZ, and the opposite is shown for MDA-MB 231 cells. Although TRD IC50 values are similar for both BC cell lines, MDA-MB 231 cells seem to be slightly more sensitive to the drug than MDA-MB 231BR ones. CPZ IC50 values also reflect what is represented in Figure 9A, being MDA-MB 231 cells two times more sensitive to the drug than MDA-MB 231BR cells.
As shown in Figure 9, all results are statistically significant, except for some low drug concentrations in MDA-MB 231BR cells. These concentrations are not high enough to cause a decrease of cell viability.
However, these results indicate that CPZ and TRD clearly reduce the viability of MDA-MB 231 and 231BR cell lines.
5.2
Effect of thioridazine and chlorpromazine on cell cycle profile in TNBC and TNBC brain metastatic cell lines
In addition to cell viability assays, effect of TRD and CPZ on cell cycle profile was also analysed. Results are represented in Figures 10-13.
In MDA-MB 231 cells, effects on cell cycle profile differ between CPZ and TRD treatment. On the one hand, and as seen in Figure 10 and Figure 11, CPZ treatment induces a G2/M arrest. This cell cycle arrest can already be observed at 24h of treatment for the higher drug concentration (5 µM), but it is clearly more evident at 48h (Figure 10). This increase in the cell population in G2/M is accompanied with a decrease of the number of cells in G0/G1, being statistically significant for CPZ 5 µM at 24h and 48h. Moreover, CPZ induces an increase in the Sub-G0/G1 cell population, indicating that the drug induces cell death at those conditions. This increment can be noticed at 24h of treatment, but it is more accentuated at 48h (Figure 11). On the other hand, TRD induces a G0/G1 arrest that can be observed at 24h of treatment with TRD 7.5 µM (Figure 10 and Figure 11). However, this arrest cannot be seen anymore at 48h of treatment, because at these conditions TRD induces cell death, as shown by the dramatic increase in the Sub- G0/G1 cell population. This increase, as expected, comes along with a decrease of cells in G0/G1 and G2/M phases (Figure 11).
23 Figure 10
Effect of CPZ and TRD on cell cycle profile in MDA-MB 231 cells
Note. MDA-MB 231 cells were treated with CPZ and TRD (concentrations indicated) for 24h and 48h, subsequently fixed and stained with PI. After that, they were analysed via flow cytometry. Every histogram represents cell count based on PI signal, delimitating four different regions, each one according to a cell cycle phase, as mentioned in the Materials and methods section. Additionally, percentage of cells distributed in each phase is also indicated. The assay was repeated 3 times, showing in this figure representative results.
CPZ 5 µM
24h 48h
TRD 7.5 µM Control MDA-MB 231
Sub-G0/G1: 3.5 % G0/G1: 57.1 % S: 9.3 % G2/M: 28.0 %
Propidium Iodide Signal
Cell Count
Sub-G0/G1: 4.6 % G0/G1: 46.2 % S: 11 % G2/M: 35.3 %
Sub-G0/G1: 9.7 % G0/G1: 29.5 % S: 14.9 % G2/M: 42.1 %
24h
Sub-G0/G1: 5.6 % G0/G1: 69.6 % S: 14.3 % G2/M: 9.6 %
Sub-G0/G1: 37.4 % G0/G1: 40.1 % S: 15.3 % G2/M: 6.3 % 48h
24 Figure 11
Effect of CPZ and TRD on cell cycle profile in MDA-MB 231 cells
Note. Bar plot representation of CPZ and TRD effects on cell cycle distribution in MDA-MB 231 cells.
Cells were treated with CPZ and TRD (concentrations indicated) for 24h and 48h, subsequently fixed and stained with PI. After that, they were analysed through flow cytometry. Each bar represents the percentage of cells in each cell cycle phase for every indicated condition. Data are the result of three independent experiments. SD for each value is also represented. Statistical significance of the differences between treatments and control is given as: *p<0.05, **p<0.01, ***p<0.001.
Sub-G0/G1 G0/G1 S G2/M
0 20 40 60 80
MDA-MB 231 treatment at 48h
Cell cycle phase
Cells in phase (%) ✱
Control CPZ 3.75 μM CPZ 5 μM
CPZ TREATMENT OF MDA-MB 231 CELLS
TRD TREATMENT OF MDA-MB 231 CELLS
Sub-G0/G1 G0/G1 S G2/M 0
20 40 60 80
24h
Cell cycle phase
Cells in phase (%)
Control CPZ 3.75 μM CPZ 5 μM
✱✱
✱
Sub-G0/G1 G0/G1 S G2/M 0
20 40 60 80
48h
Cell cycle phase
Cells in phase (%)
✱✱
Control CPZ 3.75 μM CPZ 5 μM
✱✱
✱✱✱
Sub-G0/G1 G0/G1 S G2/M 0
20 40 60 80
48h
Cell cycle phase
Cells in phase (%)
✱✱✱
Control TRD 5 μM TRD 7.5 μM
✱✱
✱
Sub-G0/G1 G0/G1 S G2/M 0
20 40 60 80
48h
Cell cycle phase
Cells in phase (%)
✱✱✱
Control TRD 5 μM TRD 7.5 μM
✱✱
✱
Sub-G0/G1 G0/G1 S G2/M 0
20 40 60 80
24h
Cell cycle phase
Cells in phase (%)
Control TRD 5 μM TRD 7.5 μM
✱✱
✱✱
✱
25 Figure 12
Effect of CPZ and TRD on cell cycle profile in MDA-MB 231BR cells
Note. MDA-MB 231BR cells were treated with CPZ and TRD (concentrations indicated) for 24h and 48h, subsequently fixed and dyed with PI. After that, they were analysed via flow cytometry. Every histogram represents cell count based on PI signal, delimitating four different regions, each one according to a cell cycle phase, as mentioned in the Materials and methods section. Additionally, percentage of cells distributed in each phase is also indicated. The assay was repeated 3 times, showing in this figure representative results.
CPZ 10 µM Control MDA-MB 231BR
TRD 7.5 µM
24h 48h
Sub-G0/G1: 5.9 % G0/G1: 58.4 % S: 9.6 % G2/M: 24.8 %
Propidium Iodide Signal
Cell Count
Sub-G0/G1: 7.6 % G0/G1: 53 % S: 8.7 % G2/M: 28.5 %
Sub-G0/G1: 21.4 % G0/G1: 29.9 % S: 5.2 % G2/M: 40.8 %
24h
Sub-G0/G1: 5.8 % G0/G1: 70.1 % S: 9.2 % G2/M: 13.7 %
48h
Sub-G0/G1: 40.6 % G0/G1: 47 % S: 6.2 % G2/M: 5.2 %
