Small Molecules Targeting Programmed Cell Death in Breast Cancer Cells

Targeted chemotherapy has become the forefront for cancer treatment in recent years. The selective and specific features allow more effective treatment with reduced side effects. Most targeted therapies, which include small molecules, act on specific molecular targets that are altered in tumour cells, mainly in cancers such as breast, lung, colorectal, lymphoma and leukaemia. With the recent exponential progress in drug development, programmed cell death, which includes apoptosis and autophagy, has become a promising therapeutic target. The research in identifying effective small molecules that target compensatory mechanisms in tumour cells alleviates the emergence of drug resistance. Due to the heterogenous nature of breast cancer, various attempts were made to overcome chemoresistance. Amongst breast cancers, triple negative breast cancer (TNBC) is of particular interest due to its heterogeneous nature in response to chemotherapy. TNBC represents approximately 15% of all breast tumours, however, and still has a poor prognosis. Unlike other breast tumours, signature targets lack for TNBCs, causing high morbidity and mortality. This review highlights several small molecules with promising preclinical data that target autophagy and apoptosis to induce cell death in TNBC cells.     

Autophagic Cell Death and Cancer

Programmed cell death (PCD) is a crucial process required for the normal development and physiology of metazoans. The three major mechanisms that induce PCD are called type I (apoptosis), type II (autophagic cell death), and type III (necrotic cell death). Dysfunctional PCD leads to diseases such as cancer and neurodegeneration. Although apoptosis is the most common form of PCD, recent studies have provided evidence that there are other forms of cell death. One of such cell death is autophagic cell death, which occurs via the activation of autophagy. The present review summarizes recent knowledge about autophagic cell death and discusses the relationship with tumorigenesis.     

Norcantharidin Induces Immunogenic Cell Death of Bladder Cancer Cells through Promoting Autophagy in Acidic Culture

The acidic tumor microenvironment stands as a major obstacle to the efficient elimination of tumor cells. Norcantharidin (NCTD) is a powerful antitumor agent with multiple bioactivities. However, the effect of NCTD under acidic conditions is still unclear. Here, we report that NCTD can efficiently kill bladder cancer (BC) cells in acidic culture, and more intriguingly, NCTD can induce immunogenic cell death (ICD), thereby promoting antitumor immunity. In NCTD-treated BC cells, the surface-exposed calreticulin (ecto-CALR) was significantly increased. Consistently, co-culture with these cells promoted dendritic cell (DC) maturation. The NCTD-induced ICD is autophagy dependent, as autophagy inhibition completely blocked the NCTD-induced ecto-CALR and DC maturation. In addition, the DC showed a distinct maturation phenotype (CD80^high CD86^low) in acidic culture, as compared to that in physiological pH (CD⁸⁰ high CD86^high). Finally, the NCTD-induced ICD was validated in a mouse model. NCTD treatment significantly increased the tumor-infiltrating T lymphocytes in MB49 bladder cancer mice. Immunizing mice with NCTD-treated MB49 cells significantly increased tumor-free survival as compared to control. These findings demonstrate that NCTD could induce ICD in an acidic environment and suggest the feasibility to combine NCTD with anticancer immunotherapy to treat BC.     

An Autophagy-Disrupting Small Molecule Promotes Cancer Cell Death via Caspase Activation

A novel autophagy inhibitor, autophazole (Atz), which promoted cancer cell death via caspase activation, is described. This compound was identified from cell-based high-content screening of an imidazole library. The results showed that Atz was internalized into lysosomes of cells where it induced lysosomal membrane permeabilization (LMP). This process generated nonfunctional autolysosomes, thereby inhibiting autophagy. In addition, Atz was found to promote LMP-mediated apoptosis. Specifically, LMP induced by Atz caused release of cathepsins from lysosomes into the cytosol. Cathepsins in the cytosol cleaved Bid to generate tBid, which subsequently activated Bax to induce mitochondrial outer membrane permeabilization (MOMP). This event led to cancer cell death via caspase activation. Overall, the findings suggest that Atz will serve as a new chemical probe in efforts aimed at gaining a better understanding of the autophagic process.     

T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death

T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.     

Immunogenic Cell Death of Breast Cancer Stem Cells Induced by an Endoplasmic Reticulum-Targeting Copper(II) Complex

Immunogenic cell death (ICD) offers a method of stimulating the immune system to attack and remove cancer cells. We report a copper(II) complex containing a Schiff base ligand and a polypyridyl ligand, 4 , capable of inducing ICD in breast cancer stem cells (CSCs). Complex 4 kills both bulk breast cancer cells and breast CSCs at sub-micromolar concentrations. Notably, 4 exhibits greater potency (one order of magnitude) towards breast CSCs than salinomycin (an established breast CSC-potent agent) and cisplatin (a clinically approved anticancer drug). Epithelial spheroid studies show that 4 is able to selectively inhibit breast CSC-enriched HMLER-shEcad spheroid formation and viability over non-tumorigenic breast MCF10 A spheroids. Mechanistic studies show that 4 operates as a Type II ICD inducer. Specifically, 4 readily enters the endoplasmic reticulum (ER) of breast CSCs, elevates intracellular reactive oxygen species (ROS) levels, induces ER stress, evokes damage-associated molecular patterns (DAMPs), and promotes breast CSC phagocytosis by macrophages. As far as we are aware, 4 is the first metal complex to induce ICD in breast CSCs and promote their engulfment by immune cells.     

Cell Death Conversion under Hypoxic Condition in Tumor Development and Therapy

Hypoxia, which is common during tumor progression, plays important roles in tumor biology. Failure in cell death in response to hypoxia contributes to progression and metastasis of tumors. On the one hand, the metabolic and oxidative stress following hypoxia could lead to cell death by triggering signal cascades, like LKB1/AMPK, PI3K/AKT/mTOR, and altering the levels of effective components, such as the Bcl-2 family, Atg and p62. On the other hand, hypoxia-induced autophagy can serve as a mechanism to turn over nutrients, so as to mitigate the adverse condition and then avoid cell death potentially. Due to the effective role of hypoxia, this review focuses on the crosstalk in cell death under hypoxia in tumor progression. Additionally, the illumination of cell death in hypoxia could shed light on the clinical applications of cell death targeted therapy.     

Effect of Exogenous pH on Cell Growth of Breast Cancer Cells

Breast cancer is the most common type of cancer in women and the most life-threatening cancer in females worldwide. One key feature of cancer cells, including breast cancer cells, is a reversed pH gradient which causes the extracellular pH of cancer cells to be more acidic than that of normal cells. Growing literature suggests that alkaline therapy could reverse the pH gradient back to normal and treat the cancer; however, evidence remains inconclusive. In this study, we investigated how different exogenous pH levels affected the growth, survival, intracellular reactive oxygen species (ROS) levels and cell cycle of triple-negative breast cancer cells from MDA-MB-231 cancer cell lines. Our results demonstrated that extreme acidic conditions (pH 6.0) and moderate to extreme basic conditions (pH 8.4 and pH 9.2) retarded cellular growth, induced cell death via necrosis and apoptosis, increased ROS levels, and shifted the cell cycle away from the G0/G1 phase. However, slightly acidic conditions (pH 6.7) increased cellular growth, decreased ROS levels, did not cause significant cell death and shifted the cell cycle from the G0/G1 phase to the G2/M phase, thereby explaining why cancer cells favored acidic conditions over neutral ones. Interestingly, our results also showed that cellular pH history did not significantly affect the subsequent growth of cells when the pH of the medium was changed. Based on these results, we suggest that controlling or maintaining an unfavorable pH (such as a slightly alkaline pH) for cancer cells in vivo could retard the growth of cancer cells or potentially treat the cancer.     

Dieckol Inhibits Autophagic Flux and Induces Apoptotic Cell Death in A375 Human Melanoma Cells via Lysosomal Dysfunction and Mitochondrial Membrane Impairment

Dieckol is a natural brown algal-derived polyphenol and its cytotoxic potential against various types of cancer cells has been studied. However, the effects of dieckol on autophagy in cancer cells remain unknown. Here, we show that dieckol inhibits the growth of A375 human melanoma cells by inducing apoptotic cell death, which is associated with lysosomal dysfunction and the inhibition of autophagic flux. Dieckol induces autophagosome accumulation by inhibiting autophagosome-lysosome fusion. Moreover, dieckol not only triggers lysosomal membrane permeabilization, followed by an increase in lysosomal pH and the inactivation of cathepsin B and D, but also causes the loss of mitochondrial membrane potential. Importantly, a cathepsin D inhibitor partially relieved dieckol-induced mitochondrial membrane impairment and caspase-mediated apoptosis. Collectively, our findings indicate that dieckol is a novel autophagy inhibitor that induces apoptosis-mediated cell death via lysosomal dysfunction and mitochondrial membrane impairment in A375 human melanoma cells. This suggests the novel potential value of dieckol as a chemotherapeutic drug candidate for melanoma treatment.     

Mechanism of Bile Acid-Induced Programmed Cell Death and Drug Discovery against Cancer: A Review

Bile acids are major signaling molecules that play a significant role as emulsifiers in the digestion and absorption of dietary lipids. Bile acids are amphiphilic molecules produced by the reaction of enzymes with cholesterol as a substrate, and they are the primary metabolites of cholesterol in the body. Bile acids were initially considered as tumor promoters, but many studies have deemed them to be tumor suppressors. The tumor-suppressive effect of bile acids is associated with programmed cell death. Moreover, based on this fact, several synthetic bile acid derivatives have also been used to induce programmed cell death in several types of human cancers. This review comprehensively summarizes the literature related to bile acid-induced programmed cell death, such as apoptosis, autophagy, and necroptosis, and the status of drug development using synthetic bile acid derivatives against human cancers. We hope that this review will provide a reference for the future research and development of drugs against cancer.     

A Phenylacetamide Resveratrol Derivative Exerts Inhibitory Effects on Breast Cancer Cell Growth

Resveratrol (RSV) is a natural compound that displays several pharmacological properties, including anti-cancer actions. However, its clinical application is limited because of its low solubility and bioavailability. Here, the antiproliferative and anti-inflammatory activity of a series of phenylacetamide RSV derivatives has been evaluated in several cancer cell lines. These derivatives contain a monosubstituted aromatic ring that could mimic the RSV phenolic nucleus and a longer flexible chain that could confer a better stability and bioavailability than RSV. Using MTT assay, we demonstrated that most derivatives exerted antiproliferative effects in almost all of the cancer cell lines tested. Among them, derivative 2, that showed greater bioavailability than RSV, was the most active, particularly against estrogen receptor positive (ER+) MCF7 and estrogen receptor negative (ER-) MDA-MB231 breast cancer cell lines. Moreover, we demonstrated that these derivatives, particularly derivative 2, were able to inhibit NO and ROS synthesis and PGE2 secretion in lipopolysaccharide (LPS)-activated U937 human monocytic cells (derived from a histiocytoma). In order to define the molecular mechanisms underlying the antiproliferative effects of derivative 2, we found that it determined cell cycle arrest at the G1 phase, modified the expression of cell cycle regulatory proteins, and ultimately triggered apoptotic cell death in both breast cancer cell lines. Taken together, these results highlight the studied RSV derivatives, particularly derivative 2, as promising tools for the development of new and more bioavailable derivatives useful in the treatment of breast cancer.     

Ym155 Induces Oxidative Stress-Mediated DNA Damage and Cell Cycle Arrest,and Causes Programmed Cell Death in Anaplastic Thyroid Cancer Cells

Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies with a median survival time of about 4 months. Currently, there is no effective treatment, and the development of new therapies is an important and urgent issue for ATC patients. YM155 is a small molecule that was identified as the top candidate in a high-throughput screen of small molecule inhibitors performed against a panel of ATC cell lines by the National Cancer Institute. However, there were no follow-up studies investigating YM155 in ATC. Here, we determined the effects of YM155 on ATC and human primary benign thyroid cell (PBTC) survival with alamarBlue assay. Our data show that YM155 inhibited proliferation of ATC cell lines while sparing normal thyroid cells, suggesting a high therapeutic window. YM155-induced DNA damage was detected by measuring phosphorylation of γ-H2AX as a marker for DNA double-strand breaks. The formamidopyrimidine-DNA glycosylase (FPG)-modified alkaline comet assay in conjunction with reactive oxygen species (ROS) assay and glutathione (GSH)/glutathione (GSSG) assay suggests that YM155-mediated oxidative stress contributes to DNA damage. In addition, we provide evidence that YM155 causes cell cycle arrest in S phase and in the G2/M transition and causes apoptosis, as seen with flow cytometry. In this study, we show for the first time the multiple effects of YM155 in ATC cells, furthering a potential therapeutic approach for ATC.     

Cell Death by Entosis: Triggers,Molecular Mechanisms and Clinical Significance

Entosis—a homotypic insertion of one cell into another, resulting in a death of the invading cell—has been described in many reports, but crucial aspects of its molecular mechanisms and clinical significance still remain controversial. While actomyosin contractility of the invading cell is very well established as a driving force in the initial phase, and autophagy induced in the outer cell is determined as the main mechanism of degradation of the inner cell, many details remain unresolved. The multitude of triggering factors and crisscrossing molecular pathways described in entosis regulation make interpretations difficult. The question of the physiological role of entosis also remains unanswered. In this review, we summarize the knowledge of molecular mechanisms and clinical data concerning entosis accumulated so far, highlighting both coherent explanations and controversies.     

6-Azauridine Induces Autophagy-Mediated Cell Death via a p53- and AMPK-Dependent Pathway

6-Azauridine (6-AZA), a pyrimidine nucleoside analogue, is known to exhibit both antitumor and antiviral activities. Although 6-AZA was discovered more than 60 years ago, the cellular effects of this compound are yet to be elucidated. Here, we report that 6-AZA regulates autophagy-mediated cell death in various human cancer cells, where 6-AZA treatment activates autophagic flux through the activation of lysosomal function. Furthermore, 6-AZA exhibited cytotoxicity in all cancer cells studied, although the mechanisms of action were diverse. In H460 cells, 6-AZA treatment induced apoptosis, and the extent of the latter could be reduced by treatment with chloroquine (CQ), a lysosomal inhibitor. However, 6-AZA treatment resulted in cell cycle arrest in H1299 cells, which could not be reversed by CQ. The cytotoxicity associated with 6-AZA treatment could be linearly correlated to the degree of autophagy-mediated cell death. In addition, we demonstrated that the cytotoxic effect of 6-AZA was dependent on AMPK and p53. These results collectively indicate that autophagy-mediated cell death triggered by 6-AZA contributes to its antitumor effect.     

Cannabidiol Antiproliferative Effect in Triple-Negative Breast Cancer MDA-MB-231 Cells Is Modulated by Its Physical State and by IGF-1

Cannabidiol (CBD) is a non-psychoactive phytocannabinoid that has been discussed for its safety and efficacy in cancer treatments. For this reason, we have inquired into its use on triple-negative human breast cancer. Analyzing the biological effects of CBD on MDA-MB-231, we have demonstrated that both CBD dosage and serum concentrations in the culture medium influence its outcomes; furthermore, light scattering studies demonstrated that serum impacts the CBD aggregation state by acting as a surfactant agent. Pharmacological studies on CBD in combination with chemotherapeutic agents reveal that CBD possesses a protective action against the cytotoxic effect exerted by cisplatin on MDA-MB-231 grown in standard conditions. Furthermore, in a low serum condition (0.5%), starting from a threshold concentration (5 µM), CBD forms aggregates, exerts cytostatic antiproliferative outcomes, and promotes cell cycle arrest activating autophagy. At doses above the threshold, CBD exerts a highly cytotoxic effect inducing bubbling cell death. Finally, IGF-1 and EGF antagonize the antiproliferative effect of CBD protecting cells from harmful consequences of CBD aggregates. In conclusion, CBD effect is strongly associated with the physical state and concentration that reaches the treated cells, parameters not taken into account in most of the research papers.     

Inhibition of Autophagy Potentiates Atorvastatin-Induced Apoptotic Cell Death in Human Bladder Cancer Cells in Vitro

Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer.     

Incorporation of Sulfonamide Moiety into Biguanide Scaffold Results in Apoptosis Induction and Cell Cycle Arrest in MCF-7 Breast Cancer Cells

Metformin, apart from its glucose-lowering properties, has also been found to demonstrate anti-cancer properties. Anti-cancer efficacy of metformin depends on its uptake in cancer cells, which is mediated by plasma membrane monoamine transporters (PMAT) and organic cation transporters (OCTs). This study presents an analysis of transporter mediated cellular uptake of ten sulfonamide-based derivatives of metformin in two breast cancer cell lines (MCF-7 and MDA-MB-231). Effects of these compounds on cancer cell growth inhibition were also determined. All examined sulfonamide-based analogues of metformin were characterized by greater cellular uptake in both MCF-7 and MDA-MB-231 cells, and stronger cytotoxic properties than those of metformin. Effective intracellular transport of the examined compounds in MCF-7 cells was accompanied by high cytotoxic activity. For instance, compound 2 with meta-methyl group in the benzene ring inhibited MCF-7 growth at micromolar range (IC₅₀ = 87.7 ± 1.18 µmol/L). Further studies showed that cytotoxicity of sulfonamide-based derivatives of metformin partially results from their ability to induce apoptosis in MCF-7 and MDA-MB-231 cells and arrest cell cycle in the G0/G1 phase. In addition, these compounds were found to inhibit cellular migration in wound healing assay. Importantly, the tested biguanides are more effective in MCF-7 cells at relatively lower concentrations than in MDA-MB-231 cells, which proves that the effectiveness of transporter-mediated accumulation in MCF-7 cells is related to biological effects, including MCF-7 cell growth inhibition, apoptosis induction and cell cycle arrest. In summary, this study supports the hypothesis that effective transporter-mediated cellular uptake of a chemical molecule determines its cytotoxic properties. These results warrant a further investigation of biguanides as putative anti-cancer agents.     

Apigenin Induces Autophagy and Cell Death by Targeting EZH2 under Hypoxia Conditions in Gastric Cancer Cells

Hypoxia is a major obstacle to gastric cancer (GC) therapy and leads to chemoresistance as GC cells are frequently exposed to the hypoxia environment. Apigenin, a flavonoid found in traditional medicine, fruits, and vegetables and an HDAC inhibitor, is a powerful anti-cancer agent against various cancer cell lines. However, detailed mechanisms involved in the treatment of GC using APG are not fully understood. In this study, we investigated the biological activity of and molecular mechanisms involved in APG-mediated treatment of GC under hypoxia. APG promoted autophagic cell death by increasing ATG5, LC3-II, and phosphorylation of AMPK and ULK1 and down-regulating p-mTOR and p62 in GC. Furthermore, our results show that APG induces autophagic cell death via the activation of the PERK signaling, indicating an endoplasmic reticulum (ER) stress response. The inhibition of ER stress suppressed APG-induced autophagy and conferred prolonged cell survival, indicating autophagic cell death. We further show that APG induces ER stress- and autophagy-related cell death through the inhibition of HIF-1α and Ezh2 under normoxia and hypoxia. Taken together, our findings indicate that APG activates autophagic cell death by inhibiting HIF-1α and Ezh2 under hypoxia conditions in GC cells.     

Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by deficiency of polycystin-1 (PC1) or polycystin-2 (PC2). Altered autophagy has recently been implicated in ADPKD progression, but its exact regulation by PC1 and PC2 remains unclear. We therefore investigated cell death and survival during nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 (PC1KO) or PC2 (PC2KO), and human urine-derived proximal tubular epithelial cells (PTEC) from early-stage ADPKD patients with PC1 mutations versus healthy individuals. Basal autophagy was enhanced in PC1-deficient cells. Similarly, following starvation, autophagy was enhanced and cell death reduced when PC1 was reduced. Autophagy inhibition reduced cell death resistance in PC1KO mIMCDs to the WT level, implying that PC1 promotes autophagic cell survival. Although PC2 expression was increased in PC1KO mIMCDs, PC2 knockdown did not result in reduced autophagy. PC2KO mIMCDs displayed lower basal autophagy, but more autophagy and less cell death following chronic starvation. This could be reversed by overexpression of PC1 in PC2KO. Together, these findings indicate that PC1 levels are partially coupled to PC2 expression, and determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress.     

Alisertib Induces Cell Cycle Arrest,Apoptosis, Autophagy and Suppresses EMT in HT29 and Caco-2 Cells

Colorectal cancer (CRC) is one of the most common malignancies worldwide with substantial mortality and morbidity. Alisertib (ALS) is a selective Aurora kinase A (AURKA) inhibitor with unclear effect and molecular interactome on CRC. This study aimed to evaluate the molecular interactome and anticancer effect of ALS and explore the underlying mechanisms in HT29 and Caco-2 cells. ALS markedly arrested cells in G₂/M phase in both cell lines, accompanied by remarkable alterations in the expression level of key cell cycle regulators. ALS induced apoptosis in HT29 and Caco-2 cells through mitochondrial and death receptor pathways. ALS also induced autophagy in HT29 and Caco-2 cells, with the suppression of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), but activation of 5′ AMP-activated protein kinase (AMPK) signaling pathways. There was a differential modulating effect of ALS on p38 MAPK signaling pathway in both cell lines. Moreover, induction or inhibition of autophagy modulated basal and ALS-induced apoptosis in both cell lines. ALS potently suppressed epithelial to mesenchymal transition (EMT) in HT29 and Caco-2 cells. Collectively, it suggests that induction of cell cycle arrest, promotion of apoptosis and autophagy, and suppression of EMT involving mitochondrial, death receptor, PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways contribute to the cancer cell killing effect of ALS on CRC cells.