Breast cancers characterized by expression of estrogen receptor-alpha  represent approximately 70% of all new cases and comprise the largest molecular subtype of this disease. Despite this high prevalence, the number of adequate experimental models of ER+ breast cancer is relatively limited. Nonetheless, these models have proved very useful in advancing understanding of how cells respond to and resist endocrine therapies, and how the ER acts as a transcription factor to regulate cell fate signaling. We discuss the primary experimental models of ER+ breast cancer including 2D and 3D cultures of established cell lines, cell line- and patient-derived xenografts, and chemically induced rodent models, with a consideration of their respective general strengths and limitations. What can and cannot be learned easily from these models is also discussed, and some observations on how these models may be used more effectively are provided. Overall, despite their limitations, the panel of models currently available has enabled major advances in the field, and these models remain central to the ability to study mechanisms of therapy action and resistance and for hypothesis testing that would otherwise be intractable or unethical in human subjects. Of the estimated > 280,000 breast cancer cases newly diagnosed in the United States annually, approximately 70% will express estrogen receptor alpha . For ER+ breast cancer patients, the recurrence risk after five years of adjuvant endocrine therapy currently ranges from ~40% (T2 N4-9 at diagnosis) to 10% (T1 N0),4. Many tumors that respond to an endocrine and/or cytotoxic therapy later recur as metastatic disease. Recurrent ER+ breast cancer is rarely cured. Interventions that interfere with ER activation have been available for over 120 years. Unfortunately, the inability of endocrine therapies to cure all ER+ breast cancers has also been a barrier to progress since the 1890s. Human breast cancer cells growing in vitro and as xenografts, chemically induced mammary tumors, and other animal models have been central to advancing our understanding of the endocrine responsiveness of ER+ breast cancer and to the development of drugs that are now in widespread use as standard of clinical care. In this review, we discuss several of the key issues associated with the use of these models in breast cancer research.Breast cancer is the most prevalent cancer and the second leading cause of cancer deaths after lung cancer in women, followed later by adrenalectomy and hypophysectomy. The first systemic intervention for ER+ breast cancer was high-dose estrogen administration, with cytotoxic chemotherapy beginning later. Unfortunately, ER+ breast cancers often exhibit a lower response rate to cytotoxic chemotherapy than other breast cancer subtypes,7. The first antiestrogen  was introduced in the early 1970s. TAM remains widely used in premenopausal women but has been supplanted by aromatase inhibitors as the first-line endocrine therapy for postmenopausal women. Differences in progression-free and disease-free survival currently favor the use of the aromatase inhibitors over TAM, as well as in the patterns of side effects, and they can help to direct an optimal therapy to individual patients. However, there is no compelling evidence of any major or widespread difference in overall survival rates between TAM, a partial ER agonist, and the aromatase inhibitors that block estradiol production,10.Early endocrine interventions for breast cancer patients included oophorectomy,11-13. Unfortunately, the optimal dosing of fulvestrant was not used in the initial clinical trials; 500 mg (not 250 mg) is now the standard dosing. Despite evidence of superiority over an aromatase inhibitor for overall survival,15, fulvestrant (500 mg) remains largely relegated to second-line therapy for postmenopausal patients. Fulvestrant requires injection as a depot that can be painful, whereas TAM and the aromatase inhibitors are given orally. It remains to be seen whether this pattern of use will persist long into the future, or whether aromatase inhibitors will be replaced by orally active ER antagonists, several of which are actively receiving clinical evaluation.The first advance beyond TAM in the context of an improvement in overall survival obtained from targeting ER was reported for the ER antagonist fulvestrant , which showed increased progression-free and overall survival activity and comparable quality of life benefit when compared with an aromatase inhibitor,17. While many CDK4/6 inhibitors are under investigation, only ribociclib  and abemaciclib (Verzenio®) in combination with an endocrine therapy have thus far shown any further increase in overall survival beyond that seen with single-agent endocrine therapy-20. The efficacy of histone deacetylase inhibitors (HDAC) in combination with endocrine therapy is also being investigated in at least 14 ongoing clinical trials, although these regimens also have shown only modest improvements in clinical benefit. Despite these advances and the notable improvements in disease-free and progression-free survival for patients with ER+ breast cancer, many patients experience distant recurrence that is eventually fatal. Clearly, there is much room for improvement. Among the most pressing issues is acquiring a greater understanding of endocrine resistance, whether de novo (some ER+ tumors never respond to an endocrine intervention) or acquired resistance (many ER+ breast cancers recur decades after an apparently successful treatment). Experimental models, both in vitro and in vivo, are likely to remain central tools in experimental approaches to acquiring this new knowledge.Most recently, combinations of an endocrine therapy (aromatase inhibitor or fulvestrant) with a CDK4/6 inhibitor have shown a significant improvement in clinical benefit relative to single agent endocrine therapy-24. All such models have limitations. The paraphrased aphorism that “All models are wrong but some models are useful”, attributed to the British statistician George E.P. Box (1919-2013), is not restricted to statistical models and is clearly also applicable to experimental models of ER+ breast cancer, as we explore in this review. The determination of which model(s) is sufficiently “useful” and which model(s) is not, along with how best these models can be used, is not necessarily trivial. Making an accurate determination is largely, but not exclusively, an issue of understanding the characteristics of the experimental model well enough to know if it is capable of allowing the investigator to test their hypothesis in a meaningful and objectively interpretable manner when used with an appropriate experimental design. Thus, the choice of model must be guided by its ability to address the question(s) at hand, rather than forcing a question upon an inadequate or inappropriate experimental model. An otherwise appropriately designed experiment forced upon a poor experimental model can produce data that may meet the expectations of the investigator yet could be misleading. Some features of this choice may be reasonably clear - testing a new antiestrogen in ER− models would be a poor choice. However, the converse assumption that testing a new endocrine intervention in any ER+ model is correct may depend on the presence or absence of ESR1 mutations (which can affect response to aromatase inhibitors), ESR2 (which can affect activity of ESR1), inherent sensitivity of the cell line selected to established antiestrogens, or other phenotypic or molecular characteristics. In this overly simple example, knowing which model(s) not to use is somewhat easier than knowing the best model(s) in which to test a new antiestrogen.Mechanism-based cancer research requires laboratory models of the human diseasein vitro and in vivo models available have generated many powerful insights. Not least among those contributions is research that supported the repurposing of the failed contraceptive TAM into one of the most successful anticancer agents for any cancer, the first molecularly targeted antineoplastic agent and the first United States FDA-approved cancer chemopreventive drug-29. Indeed, the cytotoxic, endocrine, and immunologic agents that are now used as standard of clinical care for breast cancer patients each showed safety and activity in cell culture and/or animal models at some point in their preclinical development. In this regard, the biological properties of the models appear to have been adequate to the task of early drug evaluation, despite any other cellular, genetic, and/or molecular differences among these models and apparently otherwise comparable human breast tumors.Good experimental models of ER+, endocrine-responsive breast cancer are relatively few, despite being derived from the most prevalent breast cancer subtype. Nonetheless, for many studies, whether for initial drug screening, molecular modeling, or biomarker discovery, the In vitro studies are perhaps most commonly used when there are technical or ethical reasons that the studies cannot be done in either animals or humans. For example, drug screening or mechanistic studies that may require gene knockdown, knockout, or cDNA overexpression are often best done first in vitro. Such studies can allow for multiple genetic modifications and determine their effects on responsiveness to drugs, molecular signaling, or other experimental endpoints with relative technical ease, high throughput, and low cost. The costs in time and resources to generate multiple animal models with a comparable panel of genetic modifications, and multiple drug or treatment scheduling studies, can be prohibitive. Nonetheless, preclinical animal studies are often still necessary, e.g., to support advancing a candidate drug to a first-in-human analysis. Such in vivo studies are generally left until sufficient data from appropriate in vitro experiments are available to constrain the scope and guide the design of any necessary in vivo experimentation. Importantly, this approach is also ethically sound because it can greatly limit the number of vertebrate animals required to test a more directed hypothesis.,31. For example, cellular and molecular heterogeneity are rarely synonymous. In most cell populations, some cells will be in G0 while others in G1, S, G2, or the M phase of the cell cycle, with features of their respective transcriptomes, proteomes, and metabolomes expressed differentially. If treated with a cell cycle-dependent drug, some cells will appear sensitive and some resistant. These observations provide simple examples of molecular and drug response heterogeneity in a cell population that otherwise appears to exhibit cellular homogeneity. An often-overlooked advantage of cell culture models, at least for testing some hypotheses, is their relative cellular homogeneity when compared with the mixed cell populations seen in some patient-derived xenograft (PDX) models. Relative homogeneity can reduce noise in some experimental designs or readouts. However, the type of heterogeneity can be an important factorin vitro that may (or may not) be known to affect a relevant experimental outcome; (3) apparently poor correlations of specific genetic or other features with those evident in tissue samples from patients; (4) species differences ; or (5) lack of an appropriate tumor microenvironment or supporting structure for cell lines growing in 2D culture in vitro and to a lesser degree cell lines growing in 3D culture or as xenografts. The extent to which any or all of these limitations are correct, relevant, or applicable to any given study is usually hypothesis specific. We address several of these issues, in part, elsewhere in this review. Here, we briefly comment on one of the more important concerns - the rate of false positives associated with drug screening using in vitro and in vivo models.Perhaps the most common problems leveled at tumor cell lines and animal models include: (1) the high prevalence of false positives with respect to identifying agents that are effective in preclinical studies but later show limited activity in phase II or III clinical trials; (2) long-term adaptations to growth in vitro and in vivo experimental models are used as the first line of exploration. This use reflects, among several considerations, the low cost and high throughput when used, for example, for in vitro drug discovery studies. The false positive and false negative rates of current preclinical drug screening strategies are connected because, as one rate increases, the other rate will usually decrease. For hypothesis testing, investigators seek an appropriate balance in the false positive and false negative rates. For very early drug screening, a reasonable position to take is that a high false positive rate (which identifies as active some drugs that are inactive) is more acceptable than a high false negative rate (which identifies as inactive some drugs that are active). Where the most appropriate balance between false positive and false negative outcomes lies, or how this balance should be determined, is context specific and may be affected by factors unrelated to the hypothesis, such as cost, time, availability of resources, or other constraints. Later in drug development, where an agent is finally being tested in human subjects, the costs  often dictate a balance where the likelihood of a false discovery (whether positive or negative) is as low as possible.In most cases, in vitro models and xenografts may predict poorly for subsequent clinical activity. However, given the long and multistep process of drug development, which can exceed ten years, laying the failure of an agent in phase II or III clinical trials squarely at the feet of the experimental cell lines and animal models used in the earliest stages of this process may not be entirely warranted. It is not that more and better preclinical models and tools are not needed, as they most certainly are and especially for ER+ breast cancer. Rather, focusing in this area may detract from a more objective analysis to identify flaws in every step in the process that could have a major impact on the overall process. A notably challenging issue is why many drugs that meet the requirements for successful indication of safety and efficacy in phase I and II clinical trials fail to show comparable activity in phase III trials,32. New approaches in clinical study design, such as the use of phase 0 designs, window of opportunity trials for biomarker discovery, and/or a more effective use of data from regional/global populations, continue to emerge. These and other advances may yet change the standard workflows for screening and developing some agents and eventually improve the success rate for new drugs completing phase III clinical trials.There are many reasons -50. A key consideration is an objective and informed assessment of the limitations in these models and the application of appropriate experimental designs to hypotheses that can be tested adequately within these limitations. For example, relatively few ER+ models overexpress endogenous aromatase, thus transfection with the aromatase cDNA is more widely used to create cell line models for testing drugs for aromatase inhibition-51. Cell lines used in 2D/3D culture and as xenografts in immune-deficient rodents enable studies that are intractable in more complex models. For example, many studies in human subjects have both technical and ethical limitations. Studies in humans essentially use patients or subjects as the model, for example, patients enrolled in a clinical trial are used to predict outcomes for the population at large. Patient selection is analogous, at least in principle, to the choice of a cell line or animal model with respect to designing an experiment to test a specific hypothesis. Detailed mechanistic studies can also be difficult when there is a need to deliver a specific genetic or molecular perturbation to the cancer cell components of a PDX model tumor, and to do so with broadly comparable efficiency in all appropriate cells in the xenograft. Cell lines have limitations but have been widely proven to be appropriate models for multiple aspects of breast cancer researchin vitro and in vivo, and metastasize routinely and to the most clinically relevant sites when injected as standard mammary fat pad inoculations in the most widely used immune-compromised mouse strains. Hence, there are few parental models from which to derive isogenic models of endocrine resistance. Here, we use the commonly understood definition of isogenic as it relates to cell line variants, that is, cells known to be derived from a common parental population. This definition does not require that cells be extensively sequenced to show that they have identical  genomes, which would be largely irrelevant in many cancer cell models that can acquire multiple new genetic changes quickly relative to most normal cells. Indeed, genome instability is one of the accepted hallmarks of cancer,53. As noted below, measuring a few well-defined short tandem repeats (STR) is usually adequate to establish the genetic lineage of a cell line variant as being isogenic as the term is commonly understood.Relatively few human breast cancer cell lines or PDX models express a fully endocrine sensitive phenotype in that they are ER+ and/or PR+, sensitive to all endocrine therapies currently used as standard of care in the clinic, grow both For testing some hypotheses, the lack of a large, well-characterized series of appropriate ER+ models  is a limitation in the field. Since choices are limited, how well any current ER+ breast cancer model(s) reflects the most relevant aspect(s) of the human disease to the hypothesis being tested remains an important consideration.While it has become popular in some areas to criticize established cell lines as having limited clinical relevance, such criticism often lacks an objective assessment of precisely for which characteristics of the human disease cell lines are adequate models and for which they are clearly inadequate. An inherent assumption in several such comparisons is that a single cell line, or even a panel of similar cell lines, should be a “good” reflection of what is a highly heterogeneous tumor (intratumor variability) and a highly heterogeneous disease (intertumor variability within and among currently defined molecular subtypes). Hence, a cell line may be expected to reflect some general properties shared among all tumors in the subtype of the human disease that it is believed to represent. Whether this is a reasonable assumption as such comparisons have sometimes been constructed, often in the context of genomic or transcriptomic features, may be questionable. For example, what are the most appropriate general properties, how and why were these selected, and are these properties relevant to most hypotheses that could be tested in any given cell line? Not surprisingly, cellular heterogeneous PDX models can show a “better” correlation with more features of the human disease, but whether the use of PDX models can lead to fundamentally new insights that were not already known from established cell line models remains to be determined, as is discussed elsewhere in this review.in vitro, may be present only in a small or undefined subgroup of patients, artifactual and not present in any samples from breast cancer patients, and/or relevant or irrelevant to the hypothesis being tested.One limitation in these general approaches or comparisons of experimental models to the human disease is that one question that is rarely asked effectively is: how well does any random patient sample (or model derived therefrom) also reflect the same characteristic(s) of the patient population from which the sample was drawn? This is essentially asking how good is any human sample as a model of the human disease. Hence, it is often not well established whether any deviation an individual cell line exhibits from a predetermined feature(s) of a heterogeneous patient population with which it is compared is within the range present across a larger population or other broadly similar populations of breast cancers. Some features of a cell line, perhaps gained (or lost) as a consequence of prolonged culture ,43-45,47.Some predetermined features would (or likely should) not be present in an established cell line. For example, dynamic molecular features that are essentially a reflection of changing immune cell infiltration or the presence of stromal cells that are absent in established cell line cultures will not be represented in the cell line transcriptomes. Nonetheless, at some level, it is clear that data from established cell lines maintained in 2D cell culture can exhibit significant similarities to patient samples when matched to the appropriate breast cancer subtypein vitro models, has also been raised as a limitation of established cell line models growing in vitro in 2D. As with many other issues raised in this review, care should be taken in the context of such concerns. If the goal of the study is to explore the extrinsic effects of the tumor microenvironment, then these models will have significant limitations. However, for many experimental endpoints, the extrinsic signals from the microenvironment are received, interpreted, and executed by the intrinsic machinery of the cancer cell, whether these signals are initiated by drugs, growth factors, cytokines, or other molecules derived extrinsically. Moreover, many of the most important functions of cells, such as progressing through the cell cycle, obtaining energy from glucose, or executing apoptosis, are highly conserved - not simply between normal and cancer cells of the same species and tissue but often widely across tissues and species.The absence of a 3D culture matrix in 2D cultures, as well as of a complex extracellular tumor microenvironment in 2D or 3D . Consequently, if the external (extrinsic) signals are selected, such as a drug or growth factor exposure that affects proliferation or cell survival (sensed and executed by intrinsic functions), much can be learned correctly regarding the intrinsic features that the exposure modifies from the use of 2D and 3D cultures of established cell lines. Indeed, if a human breast cancer cell line or xenograft is considered a poor model for the reasons often advanced, as noted above, the questions then arise as to what can we hope to learn about human biology from studies in fish, flies, worms, or yeast and why have we already learned so much from the study of such organisms,56.The conservation of cellular and molecular features across different species is often referred to as evolutionary parsimony, and it can explain why studying processes in quite simple species including yeast, worms, or flies can greatly advance our understanding of what happens within human cellsFor many studies, the applicability of the experimental model(s) to the clinical disease is tested indirectly. For example, a biomarker or other molecular target is identified in experimental models and the strength of a correlation between the biomarker or target and a clinically meaningful event is determined in one or more patient populations. A positive outcome - a clear and compelling correlation being found - may be interpreted that both the experimental model and the predictions associated with the molecule were “correct” (assuming that neither could have arisen by chance). A negative outcome - no correlation being found - is more difficult to interpret. For example, a lack of correlation could be because the molecule was “wrong”  and/or because the experimental model was “wrong” (the model could never be able to identify a “correct” candidate in the context of the hypothesis being tested).Mycoplasma species and cross-contamination with other cell lines. Mycoplasma is a genus of bacteria that lacks a cell wall and is often considered the simplest form of bacteria. Unfortunately, many members of this genus are resistant to common antibiotics and can be difficult to eradicate from infected cultures. Moreover, unlike contamination with other bacteria and fungi, which often turn cell culture media turbid, cell line cultures can be infected with Mycoplasma and show no immediately obvious signs of this infection. Cross-contamination from other infected cell lines is common and cultures can also be infected by researchers, since Mycoplasma are common in the human respiratory tract. Hence, infection can be prevalent; over 70% of cultures have been reported as being infected in some studies. The consequences of Mycoplasma infection can be far reaching, from altering responsiveness to steroid hormones and gene expression profiles to affecting tumorigenicity of xenografts in immune-deficient hosts. Hence, results from in vitro studies with infected cells are often unreliable and/or misleading. Unlike respiratory tract Mycoplasma, the breast microbiota interacts with breast cancer cells and may therefore impact the effectiveness of endocrine therapies,60, an underexplored research area.Two common challenges to executing reproducible cell line studies are linked to quality control issues, specifically infection with Mycoplasma infections is often difficult, the most effective strategies are: (1) to obtain and work only with cultures known to be free of contamination; (2) routine testing of cell line stock and other cultures in the laboratory; (3) to practice effective aseptic technique during all manipulations that require retaining viable cells in culture; and (4) returning to validated cell culture stocks within a reasonably short period of time  to replicate experiments. Hence, cells should be obtained from reliable sources and tested as soon as they are brought into the laboratory, with frozen stocks created only from definitively Mycoplasma-free cultures. Fortunately, there are several excellent and cost-effective tools and services available to test for Mycoplasma contamination.Since eradication of Mycoplasma infection, such contamination may not be immediately obvious. The presence of other cell lines in a cancer cell culture is reflective of the presence of multiple cell types in the tumor microenvironment and may not be visible under the microscope, particularly if the cross-contamination is from another cell line of the same or a closely related cell of origin. In addition, similar to Mycoplasma infection, the basic growth or other characteristics of the culture may not initially appear altered, although a clear morphological change in cells is often a sign of gross cross-contamination. Data from cross-contaminated cell lines can also be unreliable and/or misleading. While the problem of cell line cross-contamination is also prevalent, there are simple methods to detect the presence of even relatively few foreign cells within a culture. For many years, the standard approach was to compare the cell line karyotype against that of the original cell line or other cultures. Reporting the karyotype of new cell lines or variants of existing lines, such as drug-resistant variants derived from a sensitive parental population, was common practice.Cross-contamination by other cell lines is also highly problematic. As with  and is the current best practice for ensuring the authenticity of cell lines and the absence of cross contamination with other cells. For example, in our hands, we analyzed 16 loci in the human genome through coamplification and multicolor detection of 15 STRs and amelogenin (gender identification) and established a genetic profile for MCF-7 cells and our MCF-7 derived cell lines  and from other sources,63. Fortunately, the four basic practices noted above to reduce the risk of Mycoplasma infection also reduce the risk of cell line cross-contamination. Regular authentication solves one other common and related problem, knowing that the cell line being used is not an entirely different cell line.More recently, measuring short tandem repeat (STR) patterns has proved to exhibit both high sensitivity and high specificityll lines  with a r,65. For example, 3D matrices can be prepared from ER+, human breast cancer-associated fibroblasts, or normal breast tissue (often obtained from reduction mammoplasties). During tumor progression, stromal fibroblasts can become reprogrammed to produce an extracellular matrix altered in both physical organization and physical composition. Changes include abundant collagen I, fibronectin, desmin, smooth muscle alpha-actin, TGF-β, a specific stromal isoform of the actin binding protein palladin, and other proteins that enhance tumor growth. Studies on cell invasion often use a 3D matrix to measure the ability of cells to traverse from one side of a collagen or other matrix to the other in a Boyden chamber, Transwell, or similar apparatus.Experimental design is too complicated to cover all possible issues here. However, a few generic observations may be helpful to some readers. Most cell culture studies are done with cells growing on a 2D plastic surface, an approach that has proven useful for many types of studies for several decades. For some studies, a 3D culture matrix can offer a more physiologically relevant experimental context. However, bioavailability can be different in vitro, where the concentration of serum may affect the concentration of drug available to act. Using appropriate statistical analyses to estimate IC50 and IC80 values from dose-response data can be useful and may adjust for inherent response differences among isogenic cell lines. Recently, a growth rate inhibition endpoint has been proposed that may be less sensitive to differences in the rates of cell division among cell lines used for drug sensitivity studies. Careful selections of the most appropriate cytotoxicity assay and a series of other technical and analytical parameters,70 are critical considerations. For example, cell density can affect the rate of cell proliferation. Seeding at different cell densities can be used as a simple means to alter the proliferation rate. However, for screening agents that may affect the cell population growth rate and/or cell cycle distribution, controlling for cell density - and thus also for the proliferative capacity of the population - is a key consideration. Many cytotoxic drugs are sensitive to the cell population growth rate and/or the proportion of actively cycling cells and can exhibit different dose-response relationships when cell populations are seeded at different densities and/or growing at different rates.For many drug testing studies, using a pharmacologically based dose-response method is important, as is careful consideration of several features of experimental designThe selection of cell culture media can also affect outcomes and can be particularly important for some metabolism studies. For example, the concentration of glucose in many cell culture medium formulations is much higher than that found in normal human serum. Historically, these high concentrations were used as a matter of convenience because cancer cell lines often metabolize glucose at a high rate and can expend the glucose available so quickly that daily medium replenishment may be required. Since glucose and other key nutrients including glutamine and pyruvate are often initially present in excess in cell culture media formulations, this can affect the apparent responsiveness to agents that affect these features of cellular metabolism. Often, metabolism studies measure changes in the kinetics of substrate-product reactions governed by mass action and so may be affected when a key substrate or product is present in significant excess.For many studies, investigators choose ER− cell lines as negative controls for ER+ cells. While often a reasonable choice, there are many cellular and molecular differences between ER− models and ER+ models than cannot be explained only by the presence or absence of functional ER. Hence, care is needed when interpreting differences between ER+ and ER− models. Additional controls that are often advisable or preferable include comparing the effects of an intervention, or a change in molecular signaling, in the presence and absence of 17β-estradiol in both estrogen responsive and unresponsive isogenic ER+ variants. Showing the ability of an effect to be blocked by an appropriate antiestrogen (often a SERD rather than a SERM) in such models and experimental designs or the loss of an effect with genetic knockdown of ER can be informative. The latter approach can be challenging because some ER+ models, whether estrogen-dependent or -independent, may not tolerate loss of ER expression for a prolonged time, at least in our hands.,72. These models exhibit phenotypes, genomes, methylomes, and transcriptomes that simulate many key features of ER+ human breast tumors,43-48,73.Breast cancer is a heterogeneous group of diseases, which is broadly classified in the clinic into three major subtypes based on the expression or absence of estrogen receptor (ERα), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). Among the three subtypes, the most prevalent subtype represents 70%-80% breast cancers, which is characterized by the expression of ERα and its general dependence on estrogen for proliferation. There are several models available to study the complexity of ER+ breast cancer. Since the 1970s, breast cancer cell lines have been used to replicate and investigate key clinical and molecular features of ERα-expressing breast cancers. The three most common and widely used ER+ cell lines in the field include MCF-7, T47D, and ZR-75-1in vitro, was isolated from the pleural effusion of a Caucasian woman with metastatic breast cancer. MCF-7 cells express functional ERα. Since then, the MCF-7 cell line and its numerous variants have become standard and faithful laboratory models for many in vitro and in vivo preclinical studies. Many ERα+ breast cancer patients do not respond to or become resistant to targeted therapeutic drugs such as antiestrogens and aromatase inhibitors. In an effort to identify alterations associated with this resistance, many laboratories developed MCF-7 variants that resemble clinical characteristics of endocrine responsiveness , by selecting the cells from MCF-7-derived tumors in ovariectomized, athymic mice that did not receive any estrogenic supplementation. These cells were further inoculated in ovariectomized, athymic mice to form tumors in the absence of estrogen. The cells from this tumor were reestablished in vitro as MCF-7/LCC1 cells that showed a significantly reduced lag time in the appearance of tumors in athymic, ovariectomized mice as compared to MCF-7/MIII. Another cell line, MCF-7/LCC9, was developed in vitro by propagating MCF-7/LCC1 cells in increasing concentrations of fulvestrant, a steroidal antiestrogen anthracene (DMBA) induces mammary tumors in some rat strains, especially in Sprague-Dawley rats. While DMBA is not found in the human environment, similar PAHs are associated with breast cancer risk in humans,140, but the animals require priming (pre-treatment) with a progestin. Progestin can be given as either a single intraperitoneal injection of medroxyprogesterone acetate (MPA) or an implant that releases MPA throughout the study. MPA is given when mice are six weeks of age, with DMBA subsequently administered at a dose of 1 mg via oral gavage in weeks 7-10. About 20 weeks from the first DMBA dose, 100% of the mice exhibit at least one mammary tumor. This regimen initiates mammary tumors in C57BL/6, Balb/c, FVB, and DBA/2 mice. While other tumor types are rarely seen, some mice (< 10%) may develop tumors in lungs, digestive tract, skin, lymphoid tissue, or ovaries. The mammary tumors are mostly ER+ and both HER2 and vimentin negative,142. MPA/DMBA-initiated mouse mammary tumors are malignant and mostly resemble human luminal B breast cancers. In our hands, mammary tumors initiated by MPA/DMBA in mice do not respond to tamoxifen. Buque et al. recently reported that tamoxifen delayed the development of mammary tumors in MPA-primed, DMBA-initiated C57BL/6 mice. However, this limits the MPA/DMBA mouse model to studying factors linked to tamoxifen-induced primary prevention of mammary tumors; the model may not be well suited to studies of those factors that drive acquired resistance. Thus, the rat remains the best of the DMBA-induced rodent models in which to investigate antiestrogen resistance and local mammary tumor recurrence after an initial endocrine treatment response.DMBA can also be used to induce ER+ mammary tumors in miceC57BL/6 mouse have been used as allografts for luminal B disease given their expression of ESR2 (ERβ), although these cells also have been classified as basal-like,145. Since the stability and/or functionality of ER expression in EO771 tumors is inconsistently reported and basal-like tumors arise from allografted cells, EO771 has yet to be proven as a reliable and robustly responsive model in which to study ER+ breast cancers. In our hands, EO771 tumors were ESR1 negative and did not respond to physiological doses of TAM in vitro or in vivo (unpublished data). However, EO771 cells were highly responsive to the isoflavone genistein, a compound that activates both ESR1 and ESR2. These results are consistent with ESR2 being reported to be expressed in EO771 cells and activation of ESR2 inhibiting breast cancer cell growth. Other ER+ mouse mammary tumor cell lines have also been described, such as 67NR, but whether the resulting allografted tumors are responsive to antiestrogens has not been fully established. EO771 mouse mammary tumor cells originated from a spontaneous ER+ mammary tumor in a in vivo models for testing some hypotheses. However, the general lack of a series of genetically manipulated rodent models that exhibit a consistent and reproducible ER+ and endocrine therapy responsive phenotype remains a limitation, particularly given the high prevalence of this phenotype in patients. In addition to 3D growth of established cell lines, the generation of organoids from normal and neoplastic tissue from human subjects also offers novel opportunities to study key hypotheses in a more physiologically relevant context than simple 2D culture. Such organoids can be maintained and expanded in vitro and can be transfected with nucleic acids, enabling several lines of mechanism-based inquiry. Patient-derived organoids can naturally exhibit some cellular heterogeneity. One limitation is the ability to retain the mixed cellular organization and overall population phenotype over prolonged periods and multiple passages. When this limitation is reduced or eliminated, there is great potential for patient-derived organoids to become more widely available and more widely used. Another approach is to co-culture relatively homogeneous cultures such as cancer cells with different phenotypes, whether from established breast cancer cell lines or primary cultures, or co-cultured with cancer associated fibroblasts,148. While likely less heterogeneous than PDX models and patient-derived organoid cultures, the greater homogeneity can be useful in identifying mechanistically relevant interactions among specific, predefined cell types with less noise in the data than seen with systems where cellular heterogeneity is greater.While there are adequate models for some breast cancer subtypes, the field remains somewhat constrained by the lack of a large and diverse panel of ER+ models, particularly for the luminal A phenotype but also for the luminal B phenotype. The advent of PDX models and intraductal inoculation as a site for cell line xenografts have improved the utility of The use of established cell lines and animal models will likely remain for the foreseeable future, particularly for those studies that are technically intractable or ethically impossible in human subjects. Hence, the need for new models that can, together, capture all of the key features of the disease will also remain. The ability of experimental models of ER+ breast cancer to lead to more major discoveries in the field will continue to require a careful choice of the most appropriate model(s) and a careful design of the experiments in which the model(s) is used.


GLUT1 is a membrane associated carrier protein that functions in the physiologic transport of glucose across cell membranes. Multiple studies have shown an increased GLUT1 expression in various tumor types and a role in cancer prognosis. The aim of this study was to determine whether cutaneous sebaceous lesions have a differential expression of GLUT1 by immunohistochemistry (IHC). GLUT1 IHC was performed on excision specimens of ten cases of sebaceous carcinoma, nine of sebaceoma, ten of sebaceous adenoma, and ten of sebaceous hyperplasia. Intense, diffuse cytoplasmic staining was observed in sebaceous carcinoma. The pattern of GLUT1 staining in sebaceomas and sebaceous adenomas consisted of a gradient of intense cytoplasmic staining in the basaloid cells with a decreased intensity to membranous staining only and absent staining in mature sebaceous cells. In lesions of sebaceous hyperplasia, GLUT1 staining outlined the basal layer of each gland; cytoplasmic staining was minimal to absent. Increased cytoplasmic staining of GLUT1 may correlate with cellular metabolic and proliferative activity. GLUT1 has potential utility in differentiating sebaceous lesions. Sebaceous gland hyperplasia is commonly encountered in skin biopsy specimens while sebaceous neoplasms are relatively rare. An accurate diagnosis is imperative for treatment and follow-up, and in determining which patients should be tested for mismatch repair (MMR) deficiency, seen in Muir Torre syndrome . This syGlucose, an essential source of energy for rapidly proliferating cells, is transported through highly regulated membrane-associated proteins of the GLUT family . The GLUThe laboratory information system of the surgical pathology department of the University of Rochester Medical Center (URMC) was queried to identify cases of sebaceous hyperplasia, sebaceous adenoma, sebaceoma, and sebaceous carcinoma diagnosed from 1 January 2010 to 1 January 2020. The slides were retrieved and reviewed to verify the diagnoses and cases with sufficient tissue for additional immunohistochemical (IHC) studies were selected from each category to include ten cases each of sebaceous hyperplasia, sebaceous adenoma, and sebaceous carcinoma; only nine cases of sebaceoma were available. The patient age, gender, and tumor site were noted. In addition, the mismatch repair (MMR) protein status was reviewed in cases where immunohistochemical studies for loss of these proteins were previously conducted in the URMC laboratory. The antibody clones used for MMR evaluation were: MLH1 (M1), MSH2 (G219-1129), MSH6 (SP93), and PMS2 (A16-4).The tissue blocks for the selected cases were retrieved and slides cut for IHC staining with a monoclonal GLUT1 antibody (SPM498). The antibody is validated and currently in use for clinical cases. The results of GLUT1 IHC were evaluated using standard light microscopy.Thirty-nine sebaceous lesions were assessed. The average patient age was 66 years (range 40 to 89), 69% were male, and most lesions arose on the face or head and neck see . SebaceoGLUT1 IHC staining was distinct between the various diagnostic categories. Sebaceous carcinomas displayed intense cytoplasmic and membranous staining that was diffuse throughout the basaloid cells see . SebaceoResults for mismatch repair deficiency were available for 21 of the 39 cases. Testing had been assessed using IHC staining for proteins MLH1, PMS2, MSH2, and MSH6. Cases with the loss of at least one of these proteins were considered deficient. In sebaceous carcinomas, testing was conducted in 9/10 cases and there was a deficiency in 8/10 cases with the loss of MSH2 and/or MSH6 seen in 6/10 cases. Testing was not performed on the majority of sebaceoma cases but one case was deficient and two sebaceous adenoma cases were also deficient; protein losses were identified for MSH2 and/or MSH6 in these lesions. No cases of sebaceous hyperplasia had undergone IHC testing for MMR deficiency see . The patSebaceous adenomas, sebaceomas, and sebaceous carcinomas are neoplasms with variable proportions of immature and proliferating sebocytes. In contrast, sebaceous hyperplasia is a benign process with normal maturation in which sebocyte turnover is slowed and glands appear more numerous and crowded . Factor Testing for MMR deficiency by IHC in sebaceous neoplasms is common practice and an important step in the diagnosis of Muir-Torre syndrome ,20. As eSebaceous neoplasms and sebaceous hyperplasia display distinctive patterns of GLUT1 staining by immunohistochemistry. The patterns seen may correlate with cell immaturity and higher metabolic requirements for glucose in proliferating cells. Although GLUT1 is a membrane protein, the strong cytoplasmic staining seen in neoplastic cells demonstrates the presence of the protein in high quantities beyond the membrane, within the cytoplasm. Increased cytoplasmic staining of GLUT1 may correlate with cellular metabolic and proliferative activity in poorly differentiated, immature sebocytes. Additional immunohistochemical studies with a GLUT1 antibody will be helpful in elucidating the role of this glucose transporter in sebaceous cutaneous lesions and may aid in an accurate diagnosis. Research is currently underway to determine the utility of GLUT1 inhibitors for targeted treatment of various malignancies with multiple clinical trials in progress . In the


In general, SC are composed by a promising scaffold with great potential for the development of new drugs.Styrylchromones (SC) are a group of oxygen-containing heterocyclic compounds, which are characterized by the attachment of a styryl group to the chromone core. SC can be found in nature or can be chemically synthesized in the laboratory. As their presence in nature is scarce, the synthetic origin is the most common. Two types of SC are known: 2-styrylchromones and 3-styrylchromones. However, 2-styrylchromones are the most common, being more commonly found in nature and which chemical synthesis is more commonly described. A wide variety of SC has been described in the literature, with different substituents in different positions, the majority of which are distributed on the A- and/or B-rings. Over the years, several biological activities have been attributed to SC. This work presents a comprehensive review of the biological activities attributed to SC and their structure-activity relationship, based on a published literature search, since 1989. The following biological activities are thoroughly revised and discussed in this review: antioxidant, antiallergic, antiviral, antibacterial, antifungal, anti-inflammatory, and antitumoral, affinity and selectivity for A The chromone ring system or chromone core  . Over thH-chromen-4-one”. In the bibliographic search, 43 results were obtained, of which 4 were review articles (one of which was written in Japanese) and 39 were original articles. All the retrieved articles, written in English, from 1989 to the present (March 2021) have been analysed. From these articles, the cited references referring the biological activities of the SC were also included in this review. Furthermore, this review only includes studies with isolated compounds of synthetic and natural origin that are well characterized. Studies with SC's extracts or glycosylated derivatives were not included. Articles that do not present the methodologies used and with inconsistencies in the studied SC structures were also not included. The results presented throughout this review are presented according to the information available in the correspondent cited articles. Thus, the form of presentation of the results varies depending on the information available and may have the error associated (standard deviation or standard error of the mean) or not.The bibliographic search for this review was carried out in the PubMed database using the keywords “styrylchromone” and “styryl-4H-chromen-4-one  or 2-styryl-44-one 1, , where tne 1, 2,  are alsoHormothamnion enteromorphoides . Some of the 2-SC were more potent than the tested positive control, allopurinol (IC50 = 5.43 ± 0.08 μM). 2-SC 19 was the most active, with an IC50 value of 0.55 ± 0.03 μM, followed by 18, 14, and 17 . The structure of the most active compounds indicates that the presence of the catechol group (C-3′ and C-4′) on the B-ring of the derivatives (17-19) potentiates XO inhibition. The presence of two −OH groups linked to the A-ring also contributes to the inhibition, especially at C-5 and C-7 (19) leading to an increase in the activity when compared with the presence of just one −OH on the A-ring (17 and 18). This effect is also observed in the result obtained for 2-SC 14, which, despite having only −OH groups at C-5 and C-7 on the A-ring, proved to be a potent XO inhibitor , being more potent than the used positive control, rutin (IC50 = 3.66 ± 0.43 μM). The 2-SC from the other groups were less efficient  or completely ineffective . In summary, 2-SC from group 1 showed scavenging activity against all the detected ROS and RNS, while 2-SC from group 2 had a variable effect depending on the number and position of the −OH substituents on the A-ring, and for some reactive species, they did not show any activity. The activity of 2-SC from group 3 was generally low or inexistent. Therefore, for most of the reactive species studied, the catechol on the B-ring appears to play an important role in the scavenging activity. However, for HOCl scavenging activity, the −OH substituents on the A-ring assumed high importance, despite the existence of −OH substituents on the B-ring. The authors also tested some flavonoids structurally related to the tested 2-SC, which allowed to conclude that the styryl moiety also contributes to their observed outstanding antioxidant activity. A possible explanation for this fact might be the contribution of the styryl pattern to the stabilization of the radicals that are formed during the scavenging reactions  . Th. Th78–80 studies –39. 2-SCotype 1B  and compreported , 38.1, 5, 21, 56–58, 62, 70, and 81–83, in vitro, by a colorimetric assay, to determine the concentration of compounds that cause 50% cytotoxicity (CC50) and MNTC. Most of the tested 2-SC showed little toxicity, presenting a relatively large margin of security, at the highest concentration tested (150 μM). However, 2-SC 62, 70, and 83 showed a different toxicity profile in RAW cells, with 62 and 83 being the most toxic . Antiviral activity was evaluated by a plaque reduction assay in RAW cells. The results were presented as IC50 values and the selectivity index (SI) was also presented. 2-SC 5 and 58 were the most active  and presented high SI values . 2-SC 1, 57, 70, and 82 were less active but also demonstrated antiviral effects, and 21, 56, and 62 showed a weak anti-norovirus activity. In this study, the possibility of 2-SC 5 and 58 interfering in an early or late stage of MNV replication cycle was also evaluated. The antiviral effects of compounds decreased when they were added during the first hour of viral infection and remained when they were added after viral infection and for a continuous period of 48 h. Therefore, it was concluded that 2-SC 5 and 58 interfere more in the late stages of the virus life cycle that follow the enhancement of virus in cells. The obtained results showed that the type of substituent present at C-4′ on the B-ring influenced the potency of the anti-norovirus activity. As an example, this can be observed comparing the 4′-unsubstituted 2-SC 1 with 2-SC 21 (4′−OH substituted), 56 (4′−Cl substituted), 57 (4′−CH3 substituted), and 58 (4′−OCH3 substituted). The presence of −Cl or −OH at C-4′ on the B-ring led to a loss of the antiviral effect of 2-SC 56 or 21, respectively. 2-SC 57, with −CH3 at C-4′ on the B-ring, showed some antiviral activity but was approximately three times less potent than 2-SC 58, with −OCH3 at C-4′ on the B-ring. Thus, −OCH3 at C-4′ on the B-ring showed to be the best substituent, when there is no other substituent present in the 2-SC structure. In what concerns the C-5 substitutions on the A-ring, the presence of −OBn in 2-SC 81 led to a loss of antiviral effect, while the presence of −OH in 2-SC 5 led to one of the most active compounds. The existence of the −CH3 at C-α of the double bond of the styryl moiety led 2-SC 82 to show antiviral effect. The presence of the −OCH3 at C-3 on the C-ring appears to confer some antinorovirus activity to 2-SC 70 . In what concerns S. aureus, two strains were studied: ATCC 29212 and ATCC 43300. 2-SC 78 and 86–89 were effective against both strains for ATCC 43300 strain; however, 2-SC 92 was only effective against the ATCC 29212 strain (DIZ = 9 ± 0.12 mm). 2-SC 78 were effective against S. sciuri and S. xylosus , while 2-SC 90 was only effective against S. sciuri (DIZ = 12 ± 0.06 mm). In what concerns the Gram-negative bacteria, the studied 2-SC did not show any antibacterial activity against P. aeruginosa and K. pneumoniae. 2-SC 86–90 showed activity against E. coli strain ATCC 25922, and additionally, 2-SC 89 and 90 also showed activity against E. coli strain ATCC 35218. Analysing the structures of the tested 2-SC, in general, the 2-SC with −F on the A- and/or B-rings (78 and 86–91) were effective against a large number of studied bacterial organisms, which seems to indicate that −F plays an important role for the antibacterial activity. 2-SC 78, with only one −F at C-6 on the A-ring, showed an inhibitory effect against all the studied Gram-positive bacteria but did not show any activity against any of the Gram-negative bacteria. 2-SC 90, with −F at C-7 on the A-ring and C-4′ on the B-ring, showed antibacterial activity only against two Gram-positive bacteria (B. subtilis and S. sciuri) but was more effective than 2-SC 78 against these specific strains. Additionally, 90 also showed an inhibitory effect against both strains of E. coli (Gram-negative bacteria). 2-SC 89, with −F at C-3′ and C-5′ on the B-ring, also exhibited inhibitory effect against both strains of E. Coli and against B. subtilis and both strains of S. aureus. The presence of only one −F at C-2′ (86) or at C-3′ (87) or at C-4′ (88) on the B-ring led these 2-SC to be effective against B. subtilis and both strains of S. aureus and only one strain of E. coli (ATCC 25922). The obtained results in this study seem to indicate that the position and number of −F substituents in the A- or B-rings influence the antibacterial activity of 2-SC. 2-SC 58, 92, and 93 have no −F substituents in their structure. 2-SC 58, with −OCH3 at C-4′ on the B-ring, was only active against B. subtilis. The presence of −OCH3 at C-3′ and C-4′ on the B-ring (92) led to an inhibitory effect against B. subtilis and one strain of S. aureus (ATCC 29212). The 2-SC 93 is a methylenedioxy derivative and displayed no antibacterial activity, which apparently indicates that this group does not favour this activity .The existing works on the antibacterial activity of 2-SC include studies with different strains of Gram-positive and Gram-negative bacteria. The antibacterial activity demonstrated by 2-SC showed some variability, and it is not possible to define a clear SAR for each strain, resulting in the establishment of a generic SAR for the antibacterial activity. Thus, the above-mentioned structural characteristics that demonstrated to favour the antibacterial activity of 2-SC are summarized in Candida albicans, Aspergillus niger, Aspergillus flavus, and Penicillium chrysogenum and are below discussed. All the observed antifungal effects are summarized in Antifungal agents are used to treat fungal infections, in which the most common types are cutaneous, and ringworm infections of the skin and nails , 50. Ano94–101 , A. niger (NCIM-1196), and A. flavus (NCIM-539) by the agar dilution method. In this study, the results were presented as MIC values but the associated error was not indicated, even though the assays were performed in triplicate. 2-SC 94 and 96 were the most potent (MIC = 25 μg/mL) against C. albicans, being two times more potent that positive control, fluconazole (MIC = 50 μg/mL). For the A. niger, 2-SC 94 and 96–98 were the most potent (MIC = 25 μg/mL) and were equipotent with fluconazole. Against A. flavus, 2-SC 94, 96, and 98 were the most potent (MIC = 25 μg/mL), being equipotent with fluconazole. Analysing the substitution pattern, all the tested 2-SC have a 1,2,4-triazole ring at C-4′ on the B-ring. Interestingly, 2-SC 94, with only a 1,2,4-triazole ring at C-4′ on the B-ring as a substituent, showed an excellent antifungal activity against all the tested fungal strains. The presence of only one −CH3 at C-6 on the A-ring (95) led to a decrease in antifungal activity, when compared to 2-SC 94 and 96. The presence of a −Cl or −Br at C-6 on the A-ring  also led a decrease in antifungal potential compared to 2-SC 94. The existence of a −Cl at C-6 and C-8 on the A-ring (100) also did not favour antifungal activity. The introduction of −F at C-6 on the A-ring (98) maintained the antifungal activity against A. niger and A. flavus but decreased the potential against C. albicans, when compared to 2-SC 94. The studied 2-SC were also evaluated for their ADMET, as already mentioned in α-demethylase. The 1,2,4-triazole ring at C-4′ on the B-ring was mainly responsible for the interaction in the active site. Overall, a good binding interaction was shown between 2-SC 94–101 and the active site of fungal enzyme cytochrome P450 lanosterol 14 α-demethylase . Interestingly, 2-SC 58 was five to six times less potent at inhibiting the growth of MRC-5 cells (GI50 = 26.9 ± 3.2 μM). Regarding proliferation of peripheral human lymphocytes, 2-SC 58 (9.4–600 μM) showed to be a weak inhibitor (IC50 = 74.7 ± 4.8 μM). The cell cycle of non-tumor MRC-5 cells does not appear to be affected by 2-SC 58, since the cells presented a profile of DNA content similar to the untreated control. On the other hand, 2-SC 58 led to the hold in the G2/M phase of the cell cycle of MCF-7 cells in a concentration- and time-dependent manner. Also, for NCI-H460 cells, the effects of 2-SC 58 on the cell cycle were concentration and time dependent and led to an arrest at the G2/M phase of the cell cycle. These results led the authors to analyse the effect of 2-SC 58 (2.2–180 μM) on confluent (non-proliferative) and non-confluent  cells with the assumption that 2-SC 58 specifically affects mitosis. The results of both cell lines showed a high cellular protein content for the confluent cells, indicating that they were less sensitive to the 2-SC 58 treatment than non-confluent cells. The evaluation of cell growth by DNA synthesis for the NCI-H460 cell line indicates an inhibition of cell growth in non-confluent cells, in a concentration-dependent manner. The evaluation of the effects of 2-SC 58 (100 μM) on cytoskeleton components (actin filaments and microtubules) of MCF-7 and NCI-H460 cells during the interphase showed that the microtubule cytoskeleton appeared rich and intact in both cell lines, similar to the untreated cells of the control. Mitotic cells have undergone serious alterations, such as monopolar microtubule arrays, and rarely, normal mitotic spindles were found. For actin filaments, no significant changes were detected in both cell lines after 2-SC 58 treatment. These results suggest that 2-SC 58 specifically affects microtubules during mitosis. In the in vitro tubulin polymerization assay, 2-SC 58 (100–500 μM) led to a slight increase in tubulin polymerization, in a concentration-dependent manner, and the highest concentration tested (500 μM) resulted in the maximum polymerization plateau. In conclusion, 2-SC 58 appears to be a potential microtubule-stabilizing agent and a selective proliferation inhibitor of human tumor cell lines over normal cells, acting preferentially on proliferating cells , except for 2-SC 116, 119, 122, and 127 that were not active. Finally, 2-SC 121, 125, and 131 demonstrated good antiproliferative activity against BT483 cells, with IC50 values of 16.6 ± 2.4 μM, 15.5 ± 1.9 μM, and 16.6 ± 1.3 μM, respectively. The HeLa cell line was the most sensitive to the tested 2-SC, since most the tested 2-SC had better antiproliferative effects against this cell line when compared to the other tested cell lines, only 2-SC 119 and 124 were not active. 2-SC 129 was the most cytotoxic against the HeLa cell line (IC50 = of 4.9 ± 1.0 μM). The results obtained for the SKHep cell line showed that 2-SC 119, 120, 125, 127, and 128 were not active and 2-SC 124 and 129 were the most potent . The results of 2-SC 129 obtained for the HeLa cell line led the authors to make a mechanistic study of its antiproliferative effects. Thus, HeLa cells were treated with several concentrations of 2-SC 129 (1–10 μM), and the results showed that suppression of cell proliferation was concentration dependent. The effects of 2-SC 129 (5–20 μM) on HeLa cell morphology were also studied, and significant morphological changes have occurred, such as cell rounding and detachment from the substratum. The effects of 2-SC 116, 122, and 129 on apoptosis and cell cycle profile of HeLa cells were also evaluated to establish a relationship between the antiproliferative effect and cell cycle arrest. 2-SC 116 and 122 (20 μM) led to some cell cycle arrest in the subG1 phase, while 2-SC 129 (5–20 μM) induced cell cycle arrest in the subG1 phase in a concentration-dependent manner, which may indicate that the antiproliferative effect of 2-SC 129 observed in HeLa cells may be due to DNA fragmentation, resulting in cell death. Therefore, the effect of 2-SC 129 (5 and 10 μM) on DNA fragmentation in HeLa cells was also studied, by 4′,6-diamidino-2-phenylindole (DAPI) staining, and this fragmentation clearly occurred after treatment with 10 μM of 2-SC 129. The possibility of 2-SC 129 (10 μM) to have a microtubule-stabilizing effect was also evaluated by in vitro microtubule polymerization assay, and no effect was observed, suggesting that the antiproliferative effect on HeLa cells occurs by the other mechanisms mentioned above. In this study, the 3D-QSAR model was established by comparative molecular field analysis (CoMFA) on the BT483 cell line, to investigate how the 2-SC substituents influence the activities of the BT483 cell line. For this analysis, it was assumed that each 2-SC would adopt a conformation in its lowest energy, since the binding site is unknown. The results of the predictive model were in line with those obtained in cellular model. In what concerns the SAR, in this study, it is difficult to establish a relationship between the antiproliferative effects against the studied cell lines and the structures of the studied 2-SC, since the observed cytotoxic effects varied considerably among cell lines and regardless of the common structural characteristics among the studied 2-SC [Shaw and co-workers  synthesi116–131,  and evalied 2-SC .1, 56, 58, 59, 92, 93, 103, 104, 125, and 132–147 , HeLa, ovarian carcinoma (OVCA), SKHep, and large lung carcinoma (H460) was assessed. In addition, for 2-SC 103, growth inhibition against BT483 and colorectal adenocarcinoma (SW620) cell lines was also studied. For 2-SC 138–147, growth inhibition against AGS, PC-3, H460, and SW620 cell lines was studied. 2-SC 138 was the most active growth inhibitor (GI50 = 1.3 ± 0.07 μM) of AGS cells, followed by 2-SC 103 (GI50 = 2 ± 0.2 μM). 2-SC 1, 56, 92, 93, 104, 125, 132–134, and 143–147 did not inhibit AGS cell growth. Results of growth inhibition of 2-SC against HeLa cells showed that 2-SC 58, 59, 103, and 136 were the most potent, with GI50 varying from 6 ± 1 (103) to 9.9 ± 1 μM (136). 2-SC 56, 92, 132, and 134 did not inhibit HeLa cell growth. Against OVCA cells, 2-SC 56, 58, 103, and 135 were the most active, with GI50 values between 6 ± 1 (58) and 11 ± 1 μM (56), and 59, 93, 125, 132, 134, and 137 did not inhibit OVCA cell growth. 2-SC 58 was the most potent growth inhibitor (GI50 = 5 ± 1 μM) against the SKHep cells, and 1, 92, 93, 125, 132, and 135 did not inhibit SKHep cell growth. Results of growth inhibition of 2-SC against H460 cells showed that 2-SC 58, 103, 135, 139, and 141 presented the lowest GI50 values, varying from 6 ± 1 (103) to 10 ± 2 μM (139 and 141), and 1, 92, 93, 132, 138, 140, and 142–147 did not inhibit their growth. Only 2-SC 138 and 139 were able to inhibit the growth of PC-3 cells . For SW620 cells, 2-SC 138, 139, and 141 were able to inhibit their growth, while 2-SC 140 and 142–147 did not. Only 2-SC 103 was tested against BT483 cells and a GI50 value of 10 ± 2 μM was found. Doxorubicin was tested as a positive control against all carcinoma cell lines and showed GI50 values between 0.02 ± 0.01 and 1.76 ± 0.71 μM. The results obtained in this study reinforces the idea, previously mentioned [3 on the B-ring is related to the antitumoral activity. The results obtained for 2-SC 58 show that an −OCH3 at C-4′ on the B-ring is favourable for the inhibition of growth against the studied carcinoma cell lines, with GI50 values being relatively low. 2-SC 58 was one of the 2-SC with the best activity. 2-SC 103 that has −OCH3 at C-3′, C-4′ and C-5′ on the B-ring also presented good activity. Thus, the position that seems to most favour the activity is −OCH3 at C-4′ on the B-ring (58) or simultaneously at C-3′, C-4′, and C-5′ on the B-ring (103). In addition to −OCH3, the tested 2-SC also displayed other groups as substituents on the B-ring. Interestingly, different halogen substituents resulted in different activities: 2-SC 104, with −Br at C-4′ on the B-ring, showed growth inhibition activity against HeLa, OVCA, SKHep, and H460 cells; 2-SC 136, with −F at C-3′ and C-4′ on the B-ring, showed activity against all the studied carcinoma cells; 2-SC 56, with −Cl at C-4′ on the B-ring, demonstrated activity against OVCA, SKHep, and H460 cells. The introduction of −CF3 into C-4′ on the B-ring led 2-SC 137 to display activity against AGS, HeLa, SKHep, and H460 cells. In turn, the introduction of an −OCF3 into C-4′ on the B-ring led 2-SC 125 to only display activity against HeLa and H460 cells. 2-SC 59 has a −NO2 at C-4′ on the B-ring and was only inactive against OVCA cells and showed better activity than 2-SC 125 and 137. Since the 2-SC 103 demonstrated good activity, analogues were synthesized with −OCH3 at C-3′, C-4′ and C-5′ on the B-ring but with modifications on the A-ring (138–142). In general, the analogues (138–142) were less active than 2-SC 103, except for 2-SC 138 against AGS cells, which showed great potency. Another series of 2-SC (143–147) was also synthesized, but now with −OH at C-3′, C-4′, and C-5′ on the B-ring and modifications in several positions on the A-ring. However, none of these 2-SC showed growth inhibitory activity against the studied carcinoma cell lines, which seems to indicate that the simultaneous presence of −OH groups on the A- and B-rings does not favour this activity. The importance of double bond of the styryl moiety and the rigidity of the 2-SC structure for growth inhibitory activity was also evaluated. For this, a structure analogous to 2-SC 103 was tested, in which the double bond of the styryl moiety was hydrogenated to a single bond. This analogue and 2-SC 103 were tested against seven carcinoma cell lines . The new analogue showed a decreased activity when compared to 2-SC 103. Thus, the double bond of the styryl moiety seems to favour the growth inhibitory activity [For 2-SC entioned , 66, thaactivity .7–11  was assayed by the MTT assay, and taxol was used as positive control. In general, 2-SC 7–11 showed to be more effective against HepG2 and KB cells, being more cytotoxic for these two cell lines than for MDA-MB-231 and SMMC-7721. 2-SC 7 was the most cytotoxic against HepG2 cells (IC50 = 6.8 ± 0.3 μM), followed by 2-SC 8 (IC50 = 7.4 ± 0.1 μM). 2-SC 8 showed to be the most potent against MDA-MD-231 cells (IC50 = 8.7 ± 0.3 μM), followed by 2-SC 7 (IC50 = 11.9 ± 0.6 μM). 2-SC 9 was the most cytotoxic against KB cells (IC50 = 3.0 ± 0.2 μM) and SMMC-7721 (IC50 = 14.6 ± 0.3), and 11 was considered inactive in the last ones. Taxol was tested against the four cancer cell lines and showed IC50 values that ranged from 0.04 ± 0.002 to 1.1 ± 0.2 μM. These 2-SC have in common an −OH at C-5 and C-7 on the A-ring and at C-4′ on the B-ring, and their structure differ in the substituents present at C-6 and C-8 on the A-ring. The −CH2CH=C(CH3)2 group seems to contribute to the greater cytotoxic effect observed against HepG2 and MDA-MB-231 cells (7 and 8), while the −CH2CH(OH)CH=CH2 group seems to favour cytotoxicity against SMMC-7721 and KB cells (9 and 10). In conclusion, the presence of substituents at C-6 and C-8 on the A-ring (7–10) seems to favour the cytotoxic activity over the unsubstituted structure (11) at these positions [Yang and co-workers  isolated7–11  and repo148–151, 148 was the most cytotoxic against A549 cells (IC50 = 17.9 ± 2.9 μg/mL). 2-SC 151 showed potent cytotoxicity against HCT-15 (IC50 = 9.22 ± 1.8 μg/mL). Only 2-SC 148 showed cytotoxicity against KB cells (IC50 = 34.98 ± 6.6 μg/mL). None of the 2-SC 148–151 showed cytotoxic activity against SK-MEL-2 cells, up to a concentration of 100 μg/mL. The positive control, cisplatin, was tested against four cancer cell lines and showed IC50 values that ranged from 0.56 ± 0.12 to 1.65 ± 0.12 μg/mL. 2-SC 148–151 have in common an −OH at C-7 on the A-ring and a substituent structurally derived from lavendustin A at C-8 on the A-ring, and differ in the substituents on the B-ring. 2-SC 148 that has no substituent on the B-ring exhibited cytotoxicity against A549, HCT-15, and KB cells. In turn, 2-SC 149 that has an −OCH3 at C-4′ on the B-ring showed a decrease in the cytotoxic effect when compared to 2-SC 148, as it only showed effect against A549 and HCT-15 cells and with higher IC50 values. 2-SC 150, with −OCH3 at C-3′ and C-4′ on the B-ring, also revealed a decrease in the cytotoxic effect when compared with the activity of 2-SC 148. The presence of −Br at C-3′ on the B-ring (151) seems to favour the cytotoxic effect against HCT-15 cells, but to decrease the activity against A549 cells, when compared to 2-SC 148. In general, in the presence of a substituent structurally derived from lavendustin A at C-8 on the A-ring, the modifications at C-3′ and/or C-4′ on the B-ring did not favour the cytotoxic activity [Lee and co-workers  synthesiactivity .152–154  [152–154 (1.56–100 μM) to activate the Keap1-Nrf2 pathway was evaluated and compared to xanthohumol and sulforaphane, which were tested as positive controls, in AREc32 cells. The results were presented as the concentration required to increase luciferase activity by fivefold in relation to solvent control , but the authors did not mention the associated error. 2-SC 152–154 presented a concentration-dependent activity and showed C5 values of 2.9 μM, 18 μM, and 10.2 μM, respectively. The xanthohumol and sulforaphane presented C5 values of 7.8 μM and 4.8 μM, respectively. Thus, 2-SC 152 had better Nrf2-inducing potential than the two positive controls. 2-SC 152–154 did not show cytotoxic effects against AREc32 cells. 2-SC 152 was able to reduce the viability and proliferation , while 153 did not affect cell proliferation or induce the death of K562 cells. When the effect of 2-SC 152 on the viability of peripheral blood mononuclear cells from healthy donors was evaluated, this compound did not affect the viability of these cells, which may indicate selective toxicity for cancer cells. The obtained results revealed that the introduction of −OCH3 at C-3′ and C-4′ (153) or only at C-4′ (154) on the B-ring reduced the Nrf2-inducing activity, when compared with 2-SC 152. The same conclusion can be drawn for the antiproliferative activity against the K562 cell line, since 2-SC 153 did not have an antiproliferative effect and 2-SC 152 did. In this study, the antioxidant activity of 2-SC 152–154 was also evaluated, using the ferric-reducing antioxidant power and DPPH methods. However, these 2-SC demonstrated very low or non-existent activity, therefore this study was not mentioned in the Talhi and co-workers  evaluate152–154 . These as (K562) . The Nrfs (K562) , 72. The155–163 , melanoma, ovarian, renal, prostate, and breast. In this study, the error associated with the results was not indicated. In the first screening, 2-SC 160 showed growth inhibitory activity against four leukaemia cell lines , two non-small cell lung cancer cell lines , one CNS cancer cell line (% GI = 67%), two ovarian cancer cell lines , three renal cancer cell lines , one prostate cancer cell line (% GI = 72%), and one breast cancer cell line (% GI = 86%). 2-SC 163 inhibited the growth of five leukaemia cell lines , two non-small cell lung cancer cell lines , four colon cancer cell lines , two CNS cancer cell lines , three melanoma cell lines , four ovarian cancer cell lines , three renal cancer cell lines , two prostate cancer cell lines , and one breast cancer cell line (% GI = 70%). After this primary screening, 2-SC 160 and 163 (0.01–100 μM) were selected by the authors for an assay against cell lines of the nine types of cancer mentioned above. In this assay, GI50, the compound concentration resulting in total growth inhibition (TGI), and LC50  were calculated for each cell line. Summarizing and in general, 2-SC 163 (median GI50 = 1.9 μM) was more active than 160 (median GI50 = 2.45 μM) against the tested cell lines. The structures of these two 2-SC differ in the substituent present at C-4′ on the B-ring: 160 has a −Cl, while 163 has −N(CH3)2. These results indicate that the −N(CH3)2 at C-4′ on the B-ring favours the growth inhibitory activity in relation to the −Cl in the same position. After the results are obtained in the screening, the authors submitted the 2-SC 160 and 163 to the kinase inhibition assay to determine whether the activity of these 2-SC was related to their enzyme inhibition ability of protein kinase targets. Thus, the profile of 2-SC 160 and 163 against cyclin-dependent kinase (CDK) 2/cyclin E1, CDK4/cyclin D1, and glycogen synthase kinase 3-beta (GSK-3β) was performed [β is an enzyme that regulates numerous cellular signalling pathways and is involved in a wide range of cellular processes, such as cell cycle regulation and proliferation [160 and 163 did not inhibit the enzyme activity. 2-SC 160 and 163 showed weak inhibition against the CDK4/cyclin D protein kinase target but caused stimulation of GSK-3β activity  instead of inhibiting it. Given that 2-SC 163 was very potent in cell growth inhibition assays, it was not expected to stimulate of GSK-3β activity, but to inhibit it. Thus, this result led the authors to progress to in vivo studies with 2-SC 163 against breast cancer induced in female BALB/mice (Ehrlich solid tumor), in an attempt to correlate the high in vitro antiproliferative activity with the stimulation of GSK-3β activity. The tumor was induced and grew for two weeks, after which the mice received intraperitoneal injections of 2-SC 163 (3 mg/kg and 6 mg/kg) for five days, daily. The treatment with 2-SC 163 inhibited the growth of the tumor in a dose-dependent manner. The treatment with 2-SC 163 at low and high dose  significantly decreased the level of cyclin D1 in the tumor, whereas it increased the level of GSK-3β, when compared to the untreated group. Thus, the 2-SC 163 antitumor effect was accompanied by a significant decrease in cyclin D1 levels and a significant increase in GSK-3β levels. These results seem to indicate that tumor suppression is partially mediated by an increase in GSK-3β and a decrease in cyclin D1 levels, which suggests that cyclin D1 levels can be reduced by the activation of GSK-3β that phosphorylates cyclin D1, leading to its proteolysis [Ragab and co-workers  syntheti155–163 , with a erformed . CDKs plferation , 75. Proteolysis .164  decreased the mitochondrial membrane potential, which was more pronounced at the highest concentration. 2-SC 164 showed an inhibitory effect, in a concentration-dependent manner, on respiratory rates. Thus, the decrease in transmembrane potential induced by 164 can result from the inhibition in any respiratory complex , since it must interact with the phosphorylation system and the mitochondrial redox chain. The authors, in an attempt to elucidate the exact action of 2-SC 164 in the respiratory redox chain and in the phosphorylation system (directly on the respiratory complexes or by an uncoupler effect), evaluated the effect of 2-SC 164 on the respiratory complexes  and in the mitochondria swelling. 2-SC 164 decreased the ATP synthase and ATPase activities  and inhibited succinate dehydrogenase activity (17%). 2-SC 164 partially inhibited cytochrome c oxidase (27%), however it inhibited succinate cytochrome c reductase (≈62%). These results may indicate that 2-SC 164 essentially interacts with electron transfer at the level of complex III. Experiments in the osmotic potassium acetate medium showed that in the presence of 2-SC 164, only a small mitochondrial swelling was observed, in a concentration-dependent manner, when compared to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, used as a positive control. In conclusion, the results of this study indicate that the possible toxicological effect of 2-SC 164 may be related to the inhibition of the phosphorylation system and the electron transfer via the respiratory chain of rat liver mitochondria, especially in complex III (succinate cytochrome c reductase). The authors also suggested that 2-SC 164 may induce apoptosis through a decrease in the cellular energetic charge and promotion of the release of cytochrome c in the cytosol. Thus, the induction of mitochondrial cytochrome c release, shown in these biochemical studies, can subsequently trigger an apoptotic mechanism, an indicator of the potential cancer chemopreventive activity of 2-SC 164 [Peixoto and co-workers  studied 164  on rat l2-SC 164 .The great number of studies found in the literature about 2-SC antitumoral activity covers a wide variety of tumor cell lines, some also include normal cell lines, but not all the studies. Moreover, they comprise a wide range of 2-SC with numerous substituents in different locations, making it difficult to indicate which type of cancer is most studied and to establish a SAR. However, the above-mentioned structural characteristics that demonstrated to favour the antitumoral activity of 2-SC are summarized in 34–39, and 42–50, 50 and SI were calculated and presented. The authors mentioned the obtained CC50 values for each cell line, without the associated error, and the mean of the CC50 values obtained for the four tumor cell lines and the three normal cells, with the associated error. 3-SC 46 was the most cytotoxic against the four tumor cell lines (mean CC50 = 2.0 ± 1.2 μM), followed by 37 (mean CC50 = 6.4 ± 2.4 μM). Against normal oral cells, 3-SC 46 and 37 showed lower cytotoxicity . 3-SC 39 was also quite cytotoxic against tumor cell lines (mean CC50 = 13 ± 6.1 μM) and showed lower cytotoxicity against normal cell lines (mean CC50 = 339 ± 183 μM). Thus, these 3-SC showed high tumor specificity  and their results can be compared to those obtained for the positive controls, doxorubicin and 5-fluorouracil . Only 3-SC 48 did not show cytotoxicity against tumor cell lines nor against normal oral cell lines. In what concerns QSAR analysis, several chemical descriptors were used. The effects of functional groups on cytotoxicity of the studied 3-SC against tumor cell lines were evaluated, and the substituents −OCH3 at C-6 on the A-ring and −OH at C-4′ on the B-ring were defined to be crucial to increase the cytotoxicity against tumor cell lines. Accordingly, 3-SC 37 that has −OCH3 at C-6 on the A-ring and at C-4′ on the B-ring showed a greater cytotoxicity and tumor specificity than 3-SC 39, with −OCH3 at C-6 on the A-ring and at C-3′, C-4′ and C-5′ on the B-ring. 3-SC 38 and 39 have −OCH3 at C-6 on the A-ring and at C-3′ and C-4′ on the B-ring, but 39 also have an −OCH3 at C-5′ on the B-ring and showed better tumor specificity than 38, which indicates that this additional −OCH3 may contribute to the cytotoxic activity when in the presence of multiple −OCH3 groups on the B-ring. 3-SC 34 (with −OCH3 at C-4′ on the B-ring), 35 (with −OCH3 at C-3′ and C-4′ on the B-ring), and 36  showed low tumor specificity, which seems to indicate that the presence of −OCH3 groups only on the B-ring (without substituents at C-6 on the A-ring) decreases the cytotoxic activity against tumor cell lines. When an −OCH3 at C-6 on the A-ring is present, −Cl at C-4′ on the B-ring (43) seems to favour the cytotoxic activity against tumor cell lines over the presence of −F in the same position (42). 3-SC 44 (with −OH at C-4′ on the B-ring) and 45 (with −OH at C-3′ and C-4′ on the B-ring) have no substituents in the A-ring and showed better tumor specificity than 3-SC also without substituents in the A-ring but with −OCH3 on the B-ring, in the same positions (34–36), which may indicate that −OH on the B-ring is important for the cytotoxic activity against tumor cell lines. In 3-SC 46, −OCH3 is at C-6 on the A-ring and −OH is at C-4′ on the B-ring; while in 3-SC 48, −OCH3 is at C-4′ on the B-ring and −OH is at C-6 on the A-ring; this inversion of the substituents' positions led to the loss of 3-SC 48 cytotoxic activity. As mentioned, 3-SC 46 has an −OCH3 at C-6 on the A-ring and −OH at C-4′ on the B-ring and it showed better tumor specificity than 47, which has an additional −OH at C-3′ on the B-ring. Moreover, 3-SC 49 and 50 both have −OH at C-6 on the A-ring and C-4′ on the B-ring but 50 has an additional −OH at C-3′ on the B-ring that led to a better tumor specificity than 49. This result indicates that when C-6 on the A-ring is −OH instead of −OCH3, the addition of −OH to the B-ring can improve tumor specificity. In conclusion, 3-SC 46, with −OCH3 at C-6 on the A-ring and −OH at C-4′ on the B-ring, was the most cytotoxic and showed high tumor specificity, which indicates that these two groups, at these positions, favour the cytotoxic activity against the studied tumor cell lines [Shimada and co-workers  synthetie B-ring  seems to37, 39, and 46  than against epithelial malignant cell lines  [37, 39, and 46 demonstrated greater tumor specificity than the positive controls [46 showed high potency-selectivity to malignant tumors . To determine the minimum treatment time required for cytotoxicity induction, pulse-chase experiments were performed, where HSC-2 cells were incubated until 47 h with increasing concentrations of 37, 39, and 46 (0–1000 μM), and the relative viable cell number was determined by the MTT assay. 3-SC 46 was the most cytotoxic , followed by 37  and 39 . In this assay, it was also observed that high concentrations (100–1000 μM) of 3-SC 37 and 46 led to a cell death greater than 90%, while 3-SC 39, for the same range of concentrations, was less cytotoxic, showing a 30% cell viability. The obtained results led the authors to conclude that 3-SC 46 had the highest cytotoxicity and tumor specificity among the three 3-SC , with cytotoxicity being triggered at early 3 h after treatment. Therefore, the effects of 3-SC 46 on fine cell structures and the type of cell death were evaluated, as well as the metabolic profile induced by this 3-SC in HSC-2 cells. 3-SC 46 (3 and 10 μM) induced concentration-dependent mitochondrial vacuolization, which suggests induction of mitochondrial injury. 3-SC 46 (10 μM) suppressed autophagy and induced apoptotic cell death. In what concerns metabolomic analysis, 3-SC 46  did not significantly affect major cellular metabolic pathways, however the authors reported changes in metabolites involved in amino acid and glycerophospholipid metabolism [As described, 3-SC , and 46  showed tus study , the cytus study , the aut 6.1 μM) . Doxorub2, 40, 41, and 165–175, μM) was assessed by the MTT assay, and CC50, SI, and PSE were calculated and presented. In this study, the associated error to CC50 values was not indicated, even though the determination of the relative viable cell number was performed in triplicate. The CC50 values were presented for each cell line and the mean of the CC50 values of the four tumor cell lines, and the three normal cells were also presented, with the associated error. Doxorubicin (0.078–10 μM) and 5-fluorouracil (7.8–1000 μM) were used as positive controls. 3-SC 168 and 175 were the most cytotoxic against the tumor cell lines , followed by 169 (mean CC50 = 1.1 ± 0.31 μM). Against normal oral cells, 3-SC 168 and 175 showed low cytotoxicity , while 3-SC 169 showed high toxicity . Thus, 3-SC 168 and 175 showed high tumor specificity , being better than doxorubicin and 5-fluorouracil ; and 169 showed low tumor specificity (SI = 3.7). 3-SC 41 and 167 were also quite cytotoxic against tumor cell lines , but nevertheless also showed high cytotoxicity against normal oral cell lines . 3-SC 168 showed a high potency-selectivity expression (PSE = 49842.4), followed by 175 (PSE = 27898.1), being these values higher than those obtained for the positive controls (PSE = 24953.8 for doxorubicin and PSE = 26.2 for 5-fluorouracil). These results indicate that all tumor oral cell lines tested were more sensitive to 3-SC 168 and 175 than normal oral cells. Thus, the effects of 3-SC 168 (1–4 μM) and 175 (2–8 μM) on the apoptosis induction and growth arrest were further monitored by cell cycle analysis of HSC-2 cells. 3-SC 168 and 175 and the positive control, actinomycin D (1 μM), led to an accumulation of cell population in the subG1; however, only 3-SC 168 and 175 induced the accumulation at G2/M phase cells and induced mitotic arrest. Thus, the results demonstrated that these two 3-SC induced the apoptosis and mitotic arrest in HSC-2 cell more potently than actinomycin D. The QSAR analysis of all the tested 3-SC indicated that tumor specificity was correlated with the following descriptors: topological and 3D shape, size, electric state, aromaticity index, energy of the lowest unoccupied molecular orbital (LUMO), and partial charges. The authors also indicated that the presence of −OCH3 at C-6 on the A-ring reduced the tumor specificity and the presence of −OCH3 at C-7 on the A-ring led to an increase in tumor specificity. In fact, the 3-SC with the highest tumor specificity (168) has only −OCH3 at C-7 on the A-ring. Furthermore, the fact that 3-SC 166, with only an −OCH3 at C-6 on the A-ring, has a very low tumor specificity is an example that demonstrates that in addition to the type of substituents, their position also influences the tumor specificity. In general, the introduction of halogen atoms (−F and −Cl) at C-4′ on the B-ring (3-SC 40 and 41) reduced the tumor specificity, even in the presence of additional −OCH3 at C-7 on the A-ring (169 and 170). Unsubstituted 3-SC 2 also showed low tumor specificity. For 3-SC 165, 167, and 171, a low tumor specificity was found. These three 3-SC have in common the −N(CH3)2 group at C-4′ on the B-ring, which does not seem to favour tumor specificity. In conclusion, this study suggests that the −OCH3 group at C-7 on the A-ring favours the cytotoxic activity against the studied tumor cell lines and improves the tumor specificity of 3-SC [Takao and co-workers  synthetibicin 0.0–10 μM anAs for 2-SC, studies found in the literature on the antitumor activity of 3-SC include different tumor cell lines and a wide range of 3-SC with various substituents in different locations, making it difficult to establish a clear SAR. However, the above-mentioned structural characteristics that demonstrated to favour the antitumoral activity of 3-SC are summarized in 3 adenosine receptors, neuroprotective activity, and α-glucosidase inhibitory activity and are below discussed.In addition to the biological activities already described in the previous topics of this review, several SC have shown other less explored biological properties, such as affinity and selectivity for A3 adenosine receptors have only been reported in one study, to the best of our knowledge, in which three 2-SC were studied. Adenosine receptors are important targets for drug development as they have been associated with several biological functions, such as immune regulation and vascular function, but also with pathological processes, such as inflammatory and neurodegenerative diseases. Karton and co-workers [176–178 , acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). Years later, Takao and co-workers [1, 30, 56, 58, 88, 92, and 104–115, The potential of 2-SC as neuroprotective agents has only been studied in four studies, to the best of our knowledge. Yoon and co-workers  evaluate5  against 5  on the B-ring was also confirmed, because when they were methylated, the scavenging activity of 2-SC decreased. In contrast, the presence of −OCH3 on the B-ring (C-2′ or C-4′) seems to increase it. The presence of small alkyl groups at C-3 on the C-ring also seems to contribute to the antiallergic activity of the studied 2-SC.For the antiallergic activity, the presence of only one −CH3 at C-3 on the C-ring, and −NO2 at C-4′ on the B-ring seems to favour 2-SC antiviral activity against serotype 1B of HRV. These substituents also seem to be important for the antiviral activity of 2-SC against serotype 14 of HRV; however, the presence of −F at C-6 on the A-ring and −Cl at C-4′ on the B-ring also seems to contribute to this activity. 2-SC antiviral activity against MNV was also studied and demonstrated the importance of the presence of the −OH group at C-5 on the A-ring and the −OCH3 group at C-4′ on the B-ring for the antiviral activity against MNV. The inhibitory activity of 2-SC against the HIV-1 integrase was another of the studied antiviral effects, but only one 2-SC was evaluated, which was not very effective against the HIV-1 integrase. In what concerns 3-SC antiviral activity, there are few studies related to their antiviral properties, however in the reports found in the literature, 3-SC have shown few antiviral effects. 3-SC were studied against HIV, EV, and the serotypes 1B and 14 of HRV. The studied 3-SC did not show anti-HIV activity. In addition, it was only possible to conclude that the presence of −Cl at C-6 on the A-ring and C-4′ on the B-ring seems to play an important role for the antiviral effect against HRV 1B. The studied 3-SC showed low efficacy against EV and HRV 14, so it was not possible to establish a SAR.The antiviral potential of SC has been studied for different viruses, and the same SC have demonstrated different interactions with the different studied viruses and viruses' strains. These differences between the antiviral effects shown by SC indicate some selectivity, being an advantage for their use as antiviral agents. The analysis of the antiviral activity of 2-SC showed that it is significantly influenced by the substituents present in the structure and its location, however it is difficult to indicate in a generic way which substituents play the most important role. The presence of −Cl at C-6 on the A-ring, −OH or −OCH3 group at C-4′ on the B-ring also seems important for this activity. Summarizing, in part, the substituents that favour antibacterial activity also favour antifungal activity, which seems to indicate a weak selectivity by 2-SC.As previously mentioned, for the antibacterial and antifungal activities of SC, only 2-SC were studied and there are no reports of 3-SC with antibacterial and antifungal potential, to the best of our knowledge. The antibacterial activity of 2-SC was studied for different strains of Gram-positive and Gram-negative bacteria, with the tested 2-SC showing some variability in what concerns the results observed for each strain and between Gram-positive and negative bacteria, which makes it difficult to establish a clear SAR. Additionally, the studied 2-SC included several substituents which did not present coherent activities patterns, which also makes it difficult to establish of a specific SAR. Thus, only a generic SAR has been established for the antibacterial activity. The antibacterial activity of 2-SC seems to be influenced by the position and the number of −F substituents present on the A- or B-rings, as well as by the existence of −Cl or −OH at C-4′ on the B-ring. The C-6 on the A-ring and C-4′ on the B-ring also seem to be important substituents' positions for the antibacterial effects of 2-SC. For most 2-SC, the authors who reported their antibacterial properties, also assessed their antifungal potential. As reported for the antibacterial activity, the 2-SC studied included different substitution patterns. This fact, combined with differences in the used methodology between studies, only allowed the establishment of a generic SAR for antifungal activity. Similar to what was reported for the bacterial activity, the presence of the 1,2,4-triazole ring at C-4′ on the B-ring appears to contribute to the antifungal activity, as well as the existence of −Cl at C-4′ on the B-ring. The −OCHκB activation, and modulation of cytokines/chemokine production. Despite the reported studies being quite different and evaluating several mediators, the presence of −OH at C-3′ and C-4′ on the B-ring seems to generically favour the anti-inflammatory activity, similarly to what has been demonstrated for the antioxidant activity.Several anti-inflammatory mechanisms have been described for 2-SC, namely, inhibition of LFA-1/ICAM-1-mediated cell adhesion, interference with the arachidonic acid metabolic pathways, inhibition of NF-3 groups on the B-ring seems to play an important role in the antitumor activity performed by 2-SC, especially when the −OCH3 is at C-4′ on the B-ring. Furthermore, the simultaneous presence of −OCH3 groups at C-3′, C-4′, and C-5′ on the B-ring also seems to potentiate the antitumor effects of 2-SC. The styryl moiety seems to favour the growth inhibitory activity. The existence of −OCH3 at C-6 on the A-ring also appear to favour cytotoxic activity against tumor cell lines. All in all, the studies of the antitumoral activity of 3-SC seem to indicate that the presence of −OCH3 at C-6 or C-7 on the A-ring and a−OH at C-4′ on the B-ring favour cytotoxic activity against tumor cell lines.The antitumor potential is one of the most explored biological activities in the literature for SC. The antitumor properties of 2-SC are widely described, however there are few studies describing the antitumoral effects of 3-SC. Some of the existing studies, in addition to the evaluation of the effects of SC on tumor cell lines, also evaluate their effects on normal cells, which allows to understand whether the effects of SC are tumor selective. In addition to selectivity, it is important to know whether SC are also cytotoxic to normal cells. This fact is determinant for their use as possible antitumor agents, since the major objective is to demonstrate cytotoxicity against tumor cells, affecting as little as possible normal cells. Unfortunately, many of the studies reported did not include normal cell lines in their evaluation, making it difficult to understand if and in what extension SC are selective for tumor cell lines. In the cases where both cell types were studied, in general, SC were cytotoxic to tumor cells and did not show cytotoxicity to normal cells. These studies cover a wide variety of tumor cell lines, so it is difficult to indicate which type of cancer is most studied and to establish SAR. Nevertheless, in general, the presence of −OCHThe neuroprotective activity of SC is one of the biological activities less explored in the literature, since only the possible neuroprotective effects of three 2-SC were reported. Among these three 2-SC studied, only two showed neuroprotective properties: neuroprotective activity against glutamate-induced neurotoxicity in P12 pheochromocytoma cells and inhibition of AChE, BChE, and BACE1. Since the neuroprotective activity of 2-SC has been less studied and the effects evaluated are very different, it is difficult to identify the substituents that favour their effect, because in each study, a different 2-SC was evaluated.3 adenosine receptor, inhibitory activity of MAO-A and MAO-B, and inhibitory activity of α-glucosidase. In what concerns the affinity and selectivity of 2-SC for adenosine receptors, the presence of −OC2H5 at C-5 on the A-ring appears to favour the affinity and selectivity for the A3 adenosine receptor. For the MAO inhibitory activity, the presence of −OCH3 at C-6 on the A-ring and halogenated groups  at C-4′ on the B-ring favoured the MAO-A inhibitory activity; while the presence of −OCH3 at C-6 on the A-ring and −Cl or −Br at C-4′ on the B-ring favoured the MAO-B inhibitory activity and selectivity. The α-glucosidase inhibitory activity of 3-SC was influenced by the presence of the −OH group at C-6 on the A-ring and −OH at C-3′ and C-4′ or at C-4′ on the B-ring; however, the 3-SC with −OH at C-3′ and C-4′ on the B-ring was more potent than the 3-SC with just an −OH at C-4′ on the B-ring.As mentioned in the beginning of this section, in addition to the biological activities mentioned above, other activities have also been reported for SC, namely, affinity and selectivity for the AThis work demonstrates that SC exhibit a large variety of biological properties. Throughout this work, it can be observed that a wide variety of SC structures has already been studied, most of which with substituent variations on the A- and/or B-rings. The type of substituents in the SC structure and their location assume an essential role in each one of the activities shown. This variety, added to the experimental differences between studies, makes it difficult to establish a generic SAR for each activity. The antioxidant and antitumor activities of SC are undoubtedly the most explored ones in the literature. In conclusion, SC present a molecular scaffold that demonstrates an enormous potential for the development of new drugs envisioning the treatment of several oxidative process-related diseases, namely, inflammatory, carcinogenic, and neurodegenerative.