Dynamics of Molecular Self-Assembly of Short Peptides at Liquid-Solid Interfaces - Effect of Charged Amino Acid Point Mutations.

Self-organizing solid-binding peptides on atomically flat solid surfaces offer a unique bio/nano hybrid platform, useful for understanding the basic nature of biology/solid coupling and their practical applications. The surface behavior of peptides is determined by their molecular folding, which is influenced by various factors and is challenging to study. Here, the effect of charged amino acids is studied on the self-assembly behavior of a directed evolution selected graphite-binding dodecapeptide on graphite surface. Two mutations, M6 and M8, are designed to introduce negatively and positively charged moieties, respectively, at the anchoring domain of the wild-type (WT) peptide, affecting both binding and assembly. The questions addressed here are whether mutant peptides exhibit molecular crystal formation and demonstrate molecular recognition on the solid surface based on the specific mutations. Frequency-modulated atomic force microscopy is used for observations of the surface processes dynamically in water at molecular resolution over several hours at the ambient. The results indicate that while the mutants display distinct folding and surface behavior, each homogeneously nucleates and forms 2D self-organized patterns, akin to the WT peptide. However, their growth dynamics, domain formation, and crystalline lattice structures differ significantly. The results represent a significant step toward the rational design of bio/solid interfaces, potent facilitators of a variety of future implementations.

Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

Identification of a novel type of focal adhesion remodelling via FAK/FRNK replacement, and its contribution to cancer progression.

Numerous studies have investigated the various cellular responses against genotoxic stress, including those mediated by focal adhesions. We here identified a novel type of focal adhesion remodelling that occurs under genotoxic stress conditions, which involves the replacement of active focal adhesion kinase (FAK) with FAK-related non-kinase (FRNK). FRNK stabilized focal adhesions, leading to strong cell-matrix adhesion, and FRNK-depleted cells were easily detached from extracellular matrix upon genotoxic stress. This remodelling occurred in a wide variety of cells. In vivo, the stomachs of Frnk-knockout mice were severely damaged by genotoxic stress, highlighting the protective role of FRNK against genotoxic stress. FRNK was also found to play a vital role in cancer progression, because FRNK depletion significantly inhibited cancer dissemination and progression in a mouse cancer model. Furthermore, in human cancers, FRNK was predominantly expressed in metastatic tissues and not in primary tissues. We hence conclude that this novel type of focal adhesion remodelling reinforces cell adhesion and acts against genotoxic stress, which results in the protection of normal tissues, but in turn facilitates cancer progression.

Azithromycin, a potent autophagy inhibitor for cancer therapy, perturbs cytoskeletal protein dynamics.

BACKGROUND: Autophagy plays an important role in tumour cell growth and survival and also promotes resistance to chemotherapy. Hence, autophagy has been targeted for cancer therapy. We previously reported that macrolide antibiotics including azithromycin (AZM) inhibit autophagy in various types of cancer cells in vitro. However, the underlying molecular mechanism for autophagy inhibition remains unclear. Here, we aimed to identify the molecular target of AZM for inhibiting autophagy. METHODS: We identified the AZM-binding proteins using AZM-conjugated magnetic nanobeads for high-throughput affinity purification. Autophagy inhibitory mechanism of AZM was analysed by confocal microscopic and transmission electron microscopic observation. The anti-tumour effect with autophagy inhibition by oral AZM administration was assessed in the xenografted mice model. RESULTS: We elucidated that keratin-18 (KRT18) and α/ß-tubulin specifically bind to AZM. Treatment of the cells with AZM disrupts intracellular KRT18 dynamics, and KRT18 knockdown resulted in autophagy inhibition. Additionally, AZM treatment suppresses intracellular lysosomal trafficking along the microtubules for blocking autophagic flux. Oral AZM administration suppressed tumour growth while inhibiting autophagy in tumour tissue. CONCLUSIONS: As drug-repurposing, our results indicate that AZM is a potent autophagy inhibitor for cancer treatment, which acts by directly interacting with cytoskeletal proteins and perturbing their dynamics.

Ricolinostat enhances adavosertib­induced mitotic catastrophe in TP53­mutated head and neck squamous cell carcinoma cells.

TP53 mutation is one of the most frequent gene mutations in head and neck squamous cell carcinoma (HNSCC) and could be a potential therapeutic target. Recently, the WEE1 G2 checkpoint kinase (WEE1) inhibitor adavosertib (Adv) has attracted attention because of its selective cytotoxicity against TP53­mutated cells and has shown promising activity in early phase clinical trials. In the present study, it was demonstrated that combined treatment with Adv and a selective histone deacetylase 6 (HDAC6) inhibitor, ricolinostat (RCS), synergistically enhanced cell death induction in four out of five HNSCC cell lines with TP53 mutation (CAL27, SAS, HSC­3, and OSC­19), one HNSCC cell line with impaired TP53 function by HPV­infection (UPCI­SCC154), and TP53­knockout human lung cancer cell line (A549 TP53­KO), but not in TP53 wild­type A549 cells. Time­lapse imaging showed that RCS enhanced the Adv­induced mitotic catastrophe. Consistent with this, RCS treatment suppressed checkpoint kinase 1 (Chk1) (Ser345) phosphorylation and co­administration of RCS with Adv suppressed cyclin­dependent kinase 1 (Tyr15) phosphorylation along with increased expression of γ­H2A.X, a marker of DNA double­strand breaks in CAL27 cells. These data showed that RCS enhanced Adv­induced premature mitotic entry and cell death induction in the mitotic phase. However, although HDAC6 knockdown enhanced Adv­induced cell death with γ­H2A.X elevation, HDAC6 knockdown did not repress Chk1 phosphorylation in CAL27 cells. Our data demonstrated that the co­administration of RCS with Adv in HNSCC cells resulted in the suppression of Chk1 activity, leading to synergistically enhanced apoptosis via mitotic catastrophe in a p53­dependent manner. This enhanced cell death appeared to be partially mediated by the inhibition of HDAC6 activity by RCS.

Lysosome­targeted drug combination induces multiple organelle dysfunctions and non­canonical death in pancreatic cancer cells.

Pancreatic cancer is one of the leading causes of cancer­related mortality and has the lowest 5­year survival rate. Therefore, novel strategies are urgently required to treat pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) cells rely on enhanced lysosomal function for survival and proliferation to facilitate the degradation of contents accumulated via autophagy and macropinocytosis. Previously, we have reported that the combination of epidermal growth factor receptor/HER2 inhibitor lapatinib and sphingosine analog fingolimod (FTY720) confers a significant cytostatic effect in lung cancer cells. In the present study, the combined effects of these drugs on PDAC cell lines, BxPC­3, KP­4, PANC­1 and MIA PaCa­2, were examined. It was observed that FTY720 enhanced the lapatinib­induced cytotoxic effect and caused non­canonical and lysosome­dependent death in PDAC cells. Lapatinib and FTY720 induced lysosomal swelling and inhibited lysosomal acidification. Combination treatment with lapatinib and FTY720 increased lysosomal membrane permeability, induced mitochondrial depolarization, induced endoplasmic reticulum stress and disturbed intracellular calcium homeostasis. Additionally, the cytotoxic effect of lapatinib was enhanced by hydroxychloroquine or the CDK4/6 inhibitor abemaciclib, both of which induce lysosomal dysfunction. Collectively, these results indicated that the lysosome­targeted drug combination induces multiple organelle dysfunction and exerts a marked cytotoxic effect in PDAC cells.

Induction of synergistic non-apoptotic cell death by simultaneously targeting proteasomes with bortezomib and histone deacetylase 6 with ricolinostat in head and neck tumor cells.

Following surgery and chemoradiation, ~50% of patients with locally advanced head and neck tumors experience relapse within the first two years, with a poor prognosis. Therefore, a novel therapeutic approach is required. The aim of the present study was to investigate the effect of combination treatment with the proteasome inhibitor bortezomib (BTZ), and ricolinostat (RCS), a specific inhibitor of histone deacetylase 6 (HDAC6), on CAL27 and Detroit562 head and neck cancer cells. BTZ and RCS exhibited cytotoxicity in a dose- and time-dependent manner. Simultaneous treatment with BTZ and RCS resulted in the synergistic enhancement of non-apoptotic cell death and autophagy. The receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitor, necrostatin, but not the autophagy inhibitor, 3-methyladenine, attenuated the cytotoxicity of combined BTZ and RCS treatment. Thus, necroptosis [type-III programmed cell death (PCD)], but not autophagic cell death (type-II PCD), appeared to contribute to the pronounced cytotoxicity. However, no phosphorylation of RIPK1 or mixed lineage kinase domain-like protein was detectable in response to BTZ or RCS. Furthermore, RCS induced α-tubulin acetylation and inhibited BTZ-induced aggresome formation along with endoplasmic reticulum stress loading. Combined treatment with BTZ and RCS enhanced the production of reactive oxygen species (ROS). The ROS scavenger, N-acetyl cysteine, abrogated the increase in cytotoxicity. These results suggest the potential therapeutic value of the dual targeting of the proteasome and HDCA6 for head and neck cancers through the induction of necroptosis-like cell death along with ROS generation.

Azithromycin enhances the cytotoxicity of DNA-damaging drugs via lysosomal membrane permeabilization in lung cancer cells.

Cancer cells use autophagy for growth, survival, and cytoprotection from chemotherapy. Therefore, autophagy inhibitors appear to be good candidates for cancer treatment. Our group previously reported that macrolide antibiotics, especially azithromycin (AZM), have potent autophagy inhibitory effects, and combination treatment with tyrosine kinase inhibitors or proteasome inhibitors enhances their anti-cancer activity. In this study, we evaluated the effect of combination therapy with DNA-damaging drugs and AZM in non-small-cell lung cancer (NSCLC) cells. We found that the cytotoxic activities of DNA-damaging drugs, such as doxorubicin (DOX), etoposide, and carboplatin, were enhanced in the presence of AZM in NSCLC cell lines, whereas AZM alone exhibited almost no cytotoxicity. This enhanced cell death was dependent on wild-type-p53 status and autophagosome-forming ability because TP53 knockout (KO) and ATG5-KO cells attenuated AZM-enhanced cytotoxicity. DOX treatment upregulated lysosomal biogenesis by activating TFEB and led to lysosomal membrane damage as assessed by galectin 3 puncta assay and cytoplasmic leakage of lysosomal enzymes. In contrast, AZM treatment blocked autophagy, which resulted in the accumulation of lysosomes/autolysosomes. Thus, the effects of DOX and AZM were integrated into the marked increase in damaged lysosomes/autolysosomes, leading to prominent lysosomal membrane permeabilization (LMP) for apoptosis induction. Our data suggest that concomitant treatment with DNA-damaging drugs and AZM is a promising strategy for NSCLC treatment via pronounced LMP induction.

BRCA1 degradation in response to mitochondrial damage in breast cancer cells.

BRCA1 is a well-studied tumor suppressor involved in the homologous repair of DNA damage, whereas PINK1, a mitochondrial serine/threonine kinase, is known to be involved in mitochondrial quality control. Genetic mutations of PINK1 and Parkin cause autosomal recessive early-onset Parkinson's disease. We found that in breast cancer cells, the mitochondrial targeting reagents, which all induce mitochondrial depolarization along with PINK1 upregulation, induced proteasomal BRCA1 degradation. This BRCA1 degradation was dependent on PINK1, and BRCA1 downregulation upon mitochondrial damage caused DNA double-strand breaks. BRCA1 degradation was mediated through the direct interaction with the E3 ligase Parkin. Strikingly, BRCA1 and PINK1/Parkin expression were inversely correlated in cancerous mammary glands from breast cancer patients. BRCA1 knockdown repressed cancer cell growth, and high BRCA1 expression predicted poor relapse-free survival in breast cancer patients. These observations indicate a novel mechanism by which mitochondrial damage is transmitted to the nucleus, leading to BRCA1 degradation.

Cytonuclear Estrogen Receptor Alpha Regulates Proliferation and Migration of Endometrial Carcinoma Cells.

OBJECTIVE: The effects of estrogen on cells are mediated by the estrogen receptor α (ERα) which localizes at the peri-membrane, cytoplasm, and the nucleus of cells. Therefore, we intended to investigate how cytonuclear ERα plays its roles in different cellular activities. METHODS: We used amino acid substituted ERα that localized at the cytoplasm and nucleus but has no direct DNA-binding activities. ERα-negative endometrial carcinoma cells (ERα-) were stably transfected with plasmid of human ERα carrying a substituted phenylalanine at position 445 with alanine (ERα-F445A). Treated with 17ß-estrogen (E2) or bazedoxifene (BDF), cell proliferation, migration, and expression of kinases related to ERα signal transduction pathways were observed. RESULTS: E2 (40 nM) significantly activated proliferation in ERα-F445A cells, but not in ERα- cells. Similarly, E2 significantly activated cell migration in ERα-F445A cells, rather than that in ERα- cells. While no obvious change in the amount of the non-phosphorylated mammalian target of Rapamycin (mTOR), the expression of mTOR phosphorylated at serine 2448 decreased, which was recovered in presence of 17ß-estrogen (E2) in the ERα-F445A cells. On the other hand, the expression of focal adhesion kinase (FAK) phosphorylated at tyrosine at 297 was attenuated in the ERα-F445A cells treated with E2. CONCLUSION: It is suggested that the cytonuclear ERα-F445A induces phosphorylation of kinases in downstream pathways, which regulate cell proliferation and migration.

Macrolide antibiotics enhance the antitumor effect of lansoprazole resulting in lysosomal membrane permeabilization­associated cell death.

The proton pump inhibitor lansoprazole (LPZ) inhibits the growth of several cancer cell lines, including A549 and CAL 27. We previously reported that macrolide antibiotics such as azithromycin (AZM) and clarithromycin (CAM) potently inhibit autophagic flux and that combining AZM or CAM with the epidermal growth factor receptor inhibitors enhanced their antitumor effect against various cancer cells. In the present study, we conducted the combination treatment with LPZ and macrolide antibiotics against A549 and CAL 27 cells and evaluated cytotoxicity and morphological changes using cell proliferation and viability assays, flow cytometric analysis, immunoblotting, and morphological assessment. Combination therapy with LPZ and AZM greatly enhanced LPZ­induced cell death, whereas treatment with AZM alone exhibited negligible cytotoxicity. The observed cytotoxic effect was not mediated through apoptosis or necroptosis. Transmission electron microscopy of A549 cells treated with the LPZ + AZM combination revealed morphological changes associated with necrosis and accumulated autolysosomes with undigested contents. Furthermore, the A549 cell line with ATG5 knockout exhibited complete inhibition of autophagosome formation, which did not affect LPZ + AZM treatment­induced cytotoxicity, thus excluding the involvement of autophagy­dependent cell death in LPZ + AZM treatment­induced cell death. A549 cells treated with LPZ + AZM combination therapy retained the endosomal Alexa­dextran for extended duration as compared to untreated control cells, thus indicating impairment of lysosomal digestion. Notably, lysosomal galectin­3 puncta expression induced due to lysosomal membrane permeabilization was increased in cells treated with LPZ + AZM combination as compared to the treatment by either agent alone. Collectively, the present results revealed AZM­induced autolysosome accumulation, potentiated LPZ­mediated necrosis, and lysosomal membrane permeabilization, thus suggesting the potential clinical application of LPZ + AZM combination therapy for cancer treatment.

Sequestosome 1 (p62) accumulation in breast cancer cells suppresses progesterone receptor expression via argonaute 2.

Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression.

Abemaciclib induces atypical cell death in cancer cells characterized by formation of cytoplasmic vacuoles derived from lysosomes.

In the cell cycle, the G1 /S transition is controlled by the cyclin-dependent kinase (CDK) 4/6-cyclin D complex. Constitutive activation of CDK4/6 dysregulates G1 /S transition, leading to oncogenic transformation. We found that 3 CDK4/6 inhibitors, abemaciclib, ribociclib, and palbociclib, exerted a cytocidal effect as well as a cytostatic effect at the G1 phase in cancer cell lines, including A549 human non-small cell lung cancer cells. Among these inhibitors, abemaciclib exhibited the most potent cytotoxic effect. The cell-death phenotype induced by abemaciclib, which entailed formation of multiple cytoplasmic vacuoles, was not consistent with apoptosis or necroptosis. Abemaciclib blocked autophagic flux, resulting in accumulation of autophagosomes, however vacuole formation and cell death induced by abemaciclib were independent of autophagy. In addition, methuosis, a cell-death phenotype characterized by vacuole formation induced by excessive macropinocytosis, was excluded because the vacuoles did not incorporate fluorescent dextran. Of note, both formation of vacuoles and induction of cell death in response to abemaciclib were inhibited by vacuolar-type ATPase (V-ATPase) inhibitors such as bafilomycin A1 and concanamycin A. Live-cell imaging revealed that the abemaciclib-induced vacuoles were derived from lysosomes that expanded following acidification. Transmission electron microscopy revealed that these vacuoles contained undigested debris and remnants of organelles. Cycloheximide chase assay revealed that lysosomal turnover was blocked by abemaciclib. Furthermore, mTORC1 inhibition along with partial lysosomal membrane permeabilization occurred after abemaciclib treatment. Together, these results indicate that, in cancer cells, abemaciclib induces a unique form of cell death accompanied by swollen and dysfunctional lysosomes.

Comparison of autophagy inducibility in various tyrosine kinase inhibitors and their enhanced cytotoxicity via inhibition of autophagy in cancer cells in combined treatment with azithromycin.

Tyrosine kinase inhibitors (TKIs) induce autophagy in many types of cancer cells. We previously reported that gefitinib (GEF) and imatinib (IMA) induce autophagy in epidermal growth factor receptor (EGFR) knock-out A549 and non-BCR-ABL-expressing leukemia cell lines, respectively. This evidence suggests that TKI-induced autophagy is independent of the original target molecules. The present study compared the autophagy-inducing abilities of various TKIs, regardless of their targets, by quantitative autophagy flux assay. We established stable clones expressing the GFP-LC3-mCherry-LC3ΔG plasmid in A549, PC-9, and CAL 27 cell lines and assessed autophagy inducibility by monitoring the fluorescent ratios of GFP-LC3 to mCherry-LC3ΔG using an IncuCyte live cell imaging system during exposure to TKIs viz; GEF, osimertinib (OSI), lapatinib (LAP), lenvatinib (LEN), sorafenib (SOR), IMA, dasatinib (DAS), and tivantinib (TIV). Among these TKIs, DAS, GEF, and SOR exhibited prominent autophagy induction in A549 and PC-9 cells. In CAL 27 cells, IMA, SOR, and LEN, but not GEF, TIV, or OSI, exhibited autophagy induction. In the presence of azithromycin (AZM), which showed an inhibitory effect on autophagy flux, TKIs with prominent autophagy inducibility exhibited enhanced cytotoxicity via non-apoptotic cell death relative to effects of TKI alone. Therefore, autophagy inducibility of TKIs differed in the context of cancer cells. However, once induced, they appeared to have cytoprotective functions. Thus, blocking TKI-induced autophagy with AZM may improve the therapeutic effect of TKIs in cancer cells.

Association of BRCA Mutations and BRCAness Status With Anticancer Drug Sensitivities in Triple-Negative Breast Cancer Cell Lines

BACKGROUND: Many triple-negative breast cancers (TNBCs) show impaired breast cancer susceptibility gene I (BRCA1) function, called BRCAness. BRCAness tumors may show similar sensitivities to anticancer drugs as tumors with BRCA1 mutations. In this study, we investigated the association of BRCA mutations or BRCAness with drug sensitivities in TNBC. METHODS: BRCAness was evaluated as BRCA1-like scores, using multiplex ligation-dependent probe amplification in 12 TNBC cell lines, including four with mutations. Sensitivities to docetaxel, cisplatin, and epirubicin were compared with BRCA mutations and BRCA1-like scores. Cisplatin sensitivity was examined in BRCA1 knockdown Michigan Cancer Foundation-7 cell lines. RESULTS: Eight and four cell lines had characteristics of BRCAness and non-BRCAness, respectively. The 50% inhibitory concentration of docetaxel was higher in BRCA mutant and BRCAness cell lines than their counterparts. BRCA1-like scores showed a weak positive correlation with docetaxel sensitivity (r = 0.377; P = 0.039). Regarding cisplatin, scores were lower in BRCA mutants and BRCAness tumors than their counterparts. A negative correlation was found between BRCA1-like scores and cisplatin sensitivity (r = -0.407; P = 0.013). No differences were found for epirubicin. BRCA1 gene knockdown increased the cisplatin sensitivity of Michigan Cancer Foundation-7 cells. CONCLUSIONS: BRCA1-like scores were associated with cisplatin sensitivity and docetaxel resistance. BRCA1-like score is hence a promising indicator for estimating drug sensitivities in TNBC.

Vitamin K2 induces non-apoptotic cell death along with autophagosome formation in breast cancer cell lines.

BACKGROUND: Vitamin K2 (VK2) has been reported to induce apoptosis in many types of cancer cells including leukemia. However, there are no precise reports regarding the breast cancer cells. From the stand point of clinical implications of VK2 including chemoprevention, we investigated the effects of VK2 on breast cancer cell lines. METHODS: Breast cancer cell lines were cultured with VK2, and the cytotoxicity and cell death phenotype were examined. The HL-60 leukemia cells were used as a control for VK2-induced apoptosis. RESULTS: VK2 exhibited the cytotoxic effect, especially in triple negative breast cancer cell lines, namely, MDA-MB-231 and MDA-MB-468. However, in contrast to HL-60 cells, typical features of the cells undergoing apoptosis, such as chromatin condensation, nuclear fragments, and cleavage of caspase-3 were not detected. Transmission electron microscopy exhibited an increased number of autophagosomes/autolysosomes with plasma membrane integrity. An autophagy inhibitor, 3-methyladenine, apparently attenuated VK2-induced cytotoxicity, which indicated the involvement of autophagy-dependent cell death. Interestingly, both VK2-induced non-apoptotic cell death in MDA-MB-231 cells and VK2-induced apoptosis in HL-60 cells were suppressed in the presence of reactive oxygen species (ROS) scavengers. Therefore, ROS production by VK2 seems to be located up-stream in the molecular machinery for both the types of cell death execution. CONCLUSION: The VK2 induced non-apoptotic cell death along with autophagy, in triple negative breast cancer cell lines. Cell death phenotype induced by VK2 appears to differ among the type of cancers. This suggests the possibility of using VK2 for the breast cancer therapy.

Fingolimod sensitizes EGFR wild­type non­small cell lung cancer cells to lapatinib or sorafenib and induces cell cycle arrest.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase and mutations in this gene are major drivers of lung cancer development. EGFR tyrosine kinase inhibitors (TKIs) are standard first­line therapies for patients with advanced non­small cell lung cancer (NSCLC) with activating EGFR mutations, but are not effective in patients with wild­type EGFR. In the present study, the cytotoxic effects of various TKIs against EGFR were investigated in wild­type NSCLC cells as single treatments or in combination with Fingolimod (FTY720), which has been approved for treating multiple sclerosis and has cytotoxic effects against several tumor cell lines. It was found that the combined treatment with TKIs lapatinib (Lap) or sorafenib (Sor) and FTY720 synergistically suppressed the viability of the NSCLC cell lines A549 and H596. Additionally, FTY720 inhibited lysosomal acidification and suppressed autophagy flux. Immunoblotting and reverse transcription­quantitative polymerase chain reaction showed that FTY720 combined with Lap or Sor, enhanced endoplasmic reticulum (ER) stress loading and cell cycle arrest in A549 cells. The enhancement of ER stress loading and cell cycle arrest induced by combined treatment with Lap or Sor and FTY720, which was associated with the cytotoxicity induced by the combination of these drugs. These findings suggested that FTY720 improved TKI therapy in NSCLC patients with wild­type EGFR, by sensitizing NSCLC cells to TKIs.

Designing an effective drug combination for ER stress loading in cancer therapy using a real-time monitoring system.

Excess stress caused by accumulation of misfolded proteins inside the endoplasmic reticulum (ER) lumen can cause cells to undergo apoptosis. Misfolded proteins exported from ER to cytoplasm are ubiquitinated and mostly degraded by the proteasome, but can also be destroyed by autophagy mediated by the docking proteins p62 and NBR1. When misfolded proteins accumulate beyond the capacity of these clearance systems, they are transported to the microtubule organization center to form aggresomes, which are also degraded by autophagy. Together, these phenomena suggest the existence of a coordinated intracellular network for coping with the accumulation of misfolded proteins. Thus, rational inhibition of this network system might enhance killing of cancer cells subjected to pronounced ER stress loading. Based on this rationale, we sought to establish a quantitative assay for monitoring ER stress loading. MDA-MB231 cells stably transfected with the ERAI-Venus vector exhibited a strong XBP1 splicing signal in response to ER stress. Using the IncuCyte cell imaging system, we monitored the fluorescence intensity of XBP1-Venus, normalized against cell density, as an ER stress indicator. This parameter correlated closely with other reporters of unfolded protein responses. Assessment of the XBP1-Venus signal during exposure to various drug combinations revealed that simultaneous inhibition of the proteasome, autophagy, and aggresome formation led to more effective ER stress loading and higher cytotoxicity than inhibition of only two components. Our data suggest that this monitoring system is a useful tool for designing effective drug combinations for ER stress loading in cancer therapy.

Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines to gefitinib-mediated cytotoxicity by inducing atypical necroptosis.

The maintenance of the intracellular level of amino acids is crucial for cellular homeostasis. This is carried out via the regulation of both the influx from the extracellular environment and the recycling of intracellular resources. Since epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors, including gefitinib (GEF) have been reported to induce the apoptosis of several cancer cell lines, in the present study, we examined whether the cytotoxic effects of GEF are further enhanced under amino acid starvation (AAS) culture conditions. Under AAS culture conditions, the cell killing effect of GEF was synergistically pronounced in the EGFR-expressing cell lines, namely, CAL 27, Detroit 562, A549 and PANC-1 cells compared with those treated with either GEF or AAS alone. The addition of essential amino acids, but not non-essential amino acids to the cell culture medium resulted in the cancellation of this pronounced cytotoxicity. The knockdown of L-type amino acid transporter 1 (LAT-1) by siRNA also enhanced GEF-induced cytotoxicity. Therefore, the shortage of the intracellular amino acid pool appears to determine the sensitivity to GEF. Notably, this enhanced cytotoxicity is not mediated by the induction of apoptosis, but is accompanied by the pronounced induction of autophagy. The presence of necrostatin-1, an inhibitor of receptor-interacting serine/threonine-protein kinase 1 (RIPK­1), but not that of Z-VAD-fmk, attenuated the cytotoxic effects of GEF under AAS culture conditions. Electron microscopy demonstrated that the CAL 27 cells treated with GEF under AAS culture conditions exhibited swelling of the cytosol and organelles with an increased number of autophagosomes and autolysosomes, but without chromatin condensation and nuclear fragmentation. Autophagic cell death was excluded as the inhibition of autophagy did not attenuate the cytotoxicity. These results strongly suggest the induction of necroptosis in response to GEF under AAS culture conditions. However, we could not detect any phosphorylation of RIPK-1 and mixed lineage kinase domain like pseudokinase (MLKL), as well as any necrosome formation. Therefore, the enhanced cytotoxic effect of GEF under AAS culture conditions is thought to be mediated by atypical necroptosis.

The cyclin-dependent kinase 4/6 inhibitor, abemaciclib, exerts dose-dependent cytostatic and cytocidal effects and induces autophagy in multiple myeloma cells.

The D-type cyclin (CCND)-cyclin-dependent kinase 4/6 (CDK4/6) complex has been implicated in multiple myeloma development. We investigated the biological activity of CDK4/6 inhibitor abemaciclib on cell growth and survival in three myeloma cell lines, KMS-12-PE, RPMI 8226, and IM-9. Abemaciclib inhibited myeloma cell growth in a dose-dependent manner in all cell lines, with significant differences seen at a concentration of 320 nM. Treatment with 1 µM abemaciclib increased the fraction of cells in the G0/G1 phase and decreased the fraction in the S-G2/M phases. Further, treatment with abemaciclib at a concentration of 3.2 µM or more showed apparent cytocidal activity accompanied with cytoplasmic vacuolization against myeloma cells. Importantly, abemaciclib induced autophagy in a dose-dependent manner in all three cell lines. These results indicate that the CCND-CDK4/6 complex is closely tied to myeloma cell growth and survival.