Nano-chemical priming strategy to enhance TGF-ß resistance and anti-tumor activity of natural killer cells.

Immunotherapy based on adoptive transfer of natural killer (NK) cells is a promising strategy for circumventing the limitations of cancer treatments. However, components of the immunosuppressive tumor microenvironment (TME), such as transforming growth factor-beta (TGF-ß), compromise the therapeutic efficacy of NK cells significantly. To address these limitations, we developed a novel method of engineering NK cells for adaptive transfer. The method is based on nanogels that serve two functions: (1) they overcome the TGF-ß-mediated stress environment of the TME, and (2) they enhance the direct anti-tumor activity of NK cells. Previously, we demonstrated that cationic compounds such as 25 K branched polyethylenimine (25 K bPEI) prime NK cells, putting them in a 'ready-to-fight' state. Based on these findings, we designed nanogels that have two primary characteristics: (1) they encapsulate galunisertib (Gal), which is used clinically to inhibit TGF-ß receptor activity, thereby blocking TGF-ß signaling; and (2) they provide cells with a surface coating of 25 K bPEI. When grown in culture medium containing TGF-ß, nanogel-treated NK cells demonstrated greater migration ability, degranulation activity, and cytotoxicity towards cancer cells than untreated NK cells. Additionally, the in vivo efficacy of nanogel-treated NK cells against PC-3 xenografts was significantly greater than that of Chem_NK cells primed by 25 K bPEI alone. These findings suggest that Gal-loaded 25 K bPEI-coated nanogels exert anti-tumor effects via chemical priming, as well suppressing the effects of TGF-ß on NK cells. We also expect 25 K bPEI-based nanogels to have great potential to overcome the suppressive effects of the TME through their NK cell-priming activity and delivery of the desired chemicals.

Abnormal accumulation of extracellular vesicles in hippocampal dystrophic axons and regulation by the primary cilia in Alzheimer's disease.

Dystrophic neurites (DNs) are abnormal axons and dendrites that are swollen or deformed in various neuropathological conditions. In Alzheimer's disease (AD), DNs play a crucial role in impairing neuronal communication and function, and they may also contribute to the accumulation and spread of amyloid beta (Aß) in the brain of AD patients. However, it is still a challenge to understand the DNs of specific neurons that are vulnerable to Aß in the pathogenesis of AD. To shed light on the development of radiating DNs, we examined enriched dystrophic hippocampal axons in a mouse model of AD using a three-dimensional rendering of projecting neurons. We employed the anterograde spread of adeno-associated virus (AAV)1 and conducted proteomic analysis of synaptic compartments obtained from hippocampo-septal regions. Our findings revealed that DNs were formed due to synaptic loss at the axon terminals caused by the accumulation of extracellular vesicle (EV). Abnormal EV-mediated transport and exocytosis were identified in association with primary cilia, indicating their involvement in the accumulation of EVs at presynaptic terminals. To further address the regulation of DNs by primary cilia, we conducted knockdown of the Ift88 gene in hippocampal neurons, which impaired EV-mediated secretion of Aß and promoted accumulation of axonal spheroids. Using single-cell RNA sequencing, we identified the septal projecting hippocampal somatostatin neurons (SOM) as selectively vulnerable to Aß with primary cilia dysfunction and vesicle accumulation. Our study suggests that DNs in AD are initiated by the ectopic accumulation of EVs at the neuronal axon terminals, which is affected by neuronal primary cilia.

Development of an online discrete choice experiment to elicit preferences for tobacco treatment among college students.

OBJECTIVE: To develop an online discrete choice experiment (DCE) to elicit preferences for the format and content of tobacco treatment and determine the feasibility of the survey in face-to-face online interviews among college students. PARTICIPANTS: A convenience sample of 28 college students. METHODS: A pilot online DCE survey with sixteen choice sets was developed. The feasibility was assessed by: 1) ease of reading the survey descriptions, 2) ease of completing the DCE survey, and 3) appropriateness of the number of choice sets. Think-aloud data were analyzed to understand decision-making processes. RESULTS: All participants completed the DCE survey and reported that it was very easy to read and complete and that the number of sixteen choice sets was appropriate. Such results support the feasibility of our online DCE survey. Five decision-making strategies were identified. CONCLUSIONS: An online DCE survey administered during online interviews may replace in-person interviews for college students.

The ELK3-DRP1 axis determines the chemosensitivity of triple-negative breast cancer cells to CDDP by regulating mitochondrial dynamics.

Triple-negative breast cancer (TNBC) is the most lethal form of breast cancer. TNBC patients have higher rates of metastasis and restricted therapy options. Although chemotherapy is the conventional treatment for TNBC, the frequent occurrence of chemoresistance significantly lowers the efficacy of treatment. Here, we demonstrated that ELK3, an oncogenic transcriptional repressor that is highly expressed in TNBC, determined the chemosensitivity of two representative TNBC cell lines (MDA-MB231 and Hs578T) to cisplatin (CDDP) by regulating mitochondrial dynamics. We observed that the knockdown of ELK3 in MDA-MB231 and Hs578T rendered these cell lines more susceptible to the effects of CDDP. We further demonstrated that the chemosensitivity of TNBC cells was caused by the CDDP-mediated acceleration of mitochondrial fission, excessive mitochondrial reactive oxygen species production, and subsequent DNA damage. In addition, we identified DNM1L, a gene encoding the dynamin-related protein 1 (a major regulator of mitochondrial fission), as a direct downstream target of ELK3. Based on these results, we propose that the suppression of ELK3 expression could be used as a potential therapeutic strategy for overcoming the chemoresistance or inducing the chemosensitivity of TNBC.

Specific Signal Transduction of Constitutively Activating (D576G) and Inactivating (R476H) Mutants of Agonist-Stimulated Luteinizing Hormone Receptor in Eel.

We investigated the mechanism of signal transduction using inactivating (R476H) and activating (D576G) mutants of luteinizing hormone receptor (LHR) of eel at the conserved regions of intracellular loops II and III, respectively, naturally occurring in mammalian LHR. The expression of D576G and R476H mutants was approximately 58% and 59%, respectively, on the cell surface compared to those of eel LHR-wild type (wt). In eel LHR-wt, cAMP production increased upon agonist stimulation. Cells expressing eel LHR-D576G, a highly conserved aspartic acid residue, exhibited a 5.8-fold increase in basal cAMP response; however, the maximal cAMP response by high-agonist stimulation was approximately 0.62-fold. Mutation of a highly conserved arginine residue in the second intracellular loop of eel LHR (LHR-R476H) completely impaired the cAMP response. The rate of loss in cell-surface expression of eel LHR-wt and D576G mutant was similar to the agonist recombinant (rec)-eel LH after 30 min. However, the mutants presented rates of loss higher than eel LHR-wt did upon rec-eCG treatment. Therefore, the activating mutant constitutively induced cAMP signaling. The inactivating mutation resulted in the loss of LHR expression on the cell surface and no cAMP signaling. These data provide valuable information regarding the structure-function relationship of LHR-LH complexes.

25KDa branched polyethylenimine increases interferon-γ production in natural killer cells via improving translation efficiency.

BACKGROUND: Ex vivo cultivation is a promising strategy for increasing the number of NK cells and enhancing their antitumor activity prior to clinical application. Recent studies show that stimulation with 25KDa branched polyethylenimine (25KbPEI) generates NK cells with enhanced antitumor activity. To better understand how 25KbPEI primes NK cells, we explored the mechanism underlying increase in production of IFN-γ. METHODS: Chemical priming was performed on NK-92MI cells by incubating them with 5 µg/ml of 25KbPEI. The production of IFN-γ was evaluated by RT-qPCR, ELISA, and Flow cytometry. By evaluating the effect of pharmacological inhibition of ERK/mTOR-eIF4E signaling pathways on IFN-γ translation, the function of these signaling pathways in IFN-γ translation was examined. To comprehend the level of 25KbPEI activity on immune-related components in NK cells, RNA sequencing and proteomics analyses were conducted. RESULTS: 25KbPEI enhances the production of IFN-γ by NK cells without transcriptional activation. Activation of ERK and mTOR signaling pathways was found to be associated with 25KbPEI-mediated calcium influx in NK cells. The activation of ERK/mTOR signaling was linked to the phosphorylation of 4E-BP1, which resulted in the activation of translation initiation complex and subsequent IFN-γ translation. Analysis of RNA sequencing and proteomics data revealed that the activity of 25KbPEI to improve translation efficiency in NK cells could be extended to additional immune-related molecules. CONCLUSIONS: This study provides substantial insight into the process by which 25KbPEI primes NK cells. Our data demonstrated that the 25KbPEI mediated activation of ERK/mTOR signaling and subsequent stimulation of eIF4E is the primary mechanism by which the chemical stimulates translation of IFN-γ in NK cells. Video abstract.

ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is the most challenging subtype of breast cancer because of its aggressive behavior and the limited therapeutic strategies available. In the last decade, immunotherapy has become a promising treatment to prolong survival in advanced solid cancers including TNBC. However, the efficacy of immunotherapy in solid cancers remains limited because solid tumors contain few tumor-infiltrating lymphocytes. Here, we show that targeting an ETS transcription factor ELK3 (ELK3) recruits immune cells including natural killer (NK) cells into tumors via the chemotactic activity of chemokine. ELK3 depletion increases CXCL16 expression level and promotes NK cell cytotoxicity through CXCL16-mediated NK cell recruitment in TNBC. In silico analysis showed that ELK3 is negatively correlated with CXCL16 expression in breast cancer patient samples. Low expression of ELK3 and high expression of CXCL16 were associated with a better prognosis. Low expression of ELK3 and high expression of CXCL16 were associated with increased expression of NK cell-related genes. Our findings demonstrate that the ELK3-CXCL16 axis modulates NK cell recruitment to increase NK cell cytotoxicity, suggesting that targeting the ELK3 gene could be an adjuvant strategy for increasing the efficacy of immunotherapy in TNBC.

Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

Age of Smoking Initiation in Relation to Multiple Health Risk Factors among US Adult Smokers: National Health Interview Survey (NHIS) Data (2006-2018).

The adverse effects of multiple health risk factors have been well-documented; however, still understudied are the effects of early smoking in the context of multiple health risk factors. This study aimed to examine the role of early smoking initiation in relation to several health risk factors, including heavy drinking, physical inactivity, and obesity in later life among ever smokers in the USA. The National Health Interview Survey (NHIS) data from 2006 through 2018 were analyzed. The primary dependent variables were presence of three other risk factors: heavy drinking, physical inactivity, and obesity. The independent variable was the age of smoking initiation. Logit regression models were constructed to evaluate the association between smoking initiation and multiple health risk factors. All analyses were done in 2022. Among US adult smokers, 18.2% started smoking before age 15 (early initiators), 55.9% at ages 15-18 (middle initiators), and 25.9% at age 20 or later (late initiators). Compared to late smoking initiators, the odds of engaging in additional health risk factors increased by 37.3% among early initiators (OR = 1.373, 95% CI = 1.316, 1.432) and 7.7% among middle initiators (OR = 1.077, 95% CI= 1.041, 1.116). Additionally, current smoking was associated with higher odds (OR = 1.369, 95% CI = 1.322, 1.417) of having additional health risk factors compared to former smoking, with one exception: current smokers had lower odds of obesity (OR = 0.566, 95% CI = 0.537, 0.597). Tobacco control programs to prevent adolescents from initiating smoking may have the potential to prevent other health risk factors in adulthood.

Smoking, Stress, and Health Risk Behaviors During the Early Phase of COVID-19 Lockdowns Among Residents of Flint, Michigan.

Food insecurity and smoking commonly co-occur in underserved populations, exacerbating health disparities. This study assesses smoking and its associations with mental health and health risk behaviors among residents of Flint, Michigan in the early phase of COVID lockdowns. The survey captured smoking status, mental health indicators, and health risk behaviors among 106 who had financial needs and limited mobility and received community nutritional services. Comparisons by demographics showed that those who lived alone, used marijuana, and had frequent alcohol drinking tended to smoke more than their counterparts. Results from logistic regression showed that those with high levels of stress, marijuana use, and alcohol consumption were more likely to smoke. Living alone was associated with smoking and health risk behaviors. This research suggests the need for smoking prevention and community health practices addressing stress, health risk behaviors, and circumstances that might drive the association with living alone such as loneliness.

ELK3-ID4 axis governs the metastatic features of triple negative breast cancer.

Purpose: Cancer cell metastasis is a multistep process, and the mechanism underlying extravasation remains unclear. ELK3 is a transcription factor that plays a crucial role in regulating various cellular processes, including cancer metastasis. Based on the finding that ELK3 promotes the metastasis of triple-negative breast cancer (TNBC), we investigated whether ELK3 regulates the extravasation of TNBC by forming the ELK3-ID4 axis. ID4 functions as a transcriptional regulator that interacts with other transcription factors, inhibiting their activity and subsequently influencing various biological processes associated with cell differentiation, survival, growth, and metastasis. Methods: We assessed the correlation between the expression of ELK3 and that of ID4 in TNBCs using bioinformatics analyses, QRT-PCR, western blot analysis, luciferase reporter assays, and chromatin immunoprecipitation. Migration, adhesion, invasion, and lung metastasis assays were employed to determine whether the ELK3-ID4 axis regulates the metastatic features of TNBC. Results: We found that ELK3 binds directly to a binding motif close to the ID4 promoter to repress promoter activity. The expression of E-cadherin in TNBC was regulated by the ELK3-ID4 axis. In vitro and in vivo analyses showed that inhibiting ID4 expression in ELK3-knockdown MDA-MB-231 (ELK3KD) cells restored the ability to extravasate and metastasize. Conclusion: The results indicate that the ELK3 regulates ID4 promoter activity, and that the ELK3-ID4 axis regulates the metastatic characteristics of TNBC cells. Additionally, the data suggest that the ELK3-ID4 axis regulates metastasis of TNBCs by modulating expression of E-cadherin.

Chemical priming of natural killer cells with branched polyethylenimine for cancer immunotherapy.

BACKGROUND: Due to their powerful immune surveillance activity and ability to kill and clear cancer cells, natural killer (NK) cells are an emerging anticancer immunotherapeutic agent. Therefore, there is much interest in developing efficient technologies that further enhance the therapeutic antitumor efficacy of NK cells. METHODS: To produce chemically primed NK cells, we screened polymers with various electric charges and examined their ability to enhance the cytotoxicity of NK cells. The effect of primary amine and electric charges of 25 kDa branched polyethylenimine (25KbPEI) was investigated by fluorination of the chemical. The role of 25KbPEI in determining the major priming mechanism was investigated in terms of calcium influx into NK cells. In vivo therapeutic efficacy of chemically primed NK cells was evaluated against solid tumor mouse model of triple negative breast and ovarian cancers. RESULTS: Chem_NK that was produced by the priming activity of 25KbPEI showed potent antitumor activity to various cancer cells. Chem_NK showed an activated phenotype, which manifests as increased expression of activating/adhesion/chemokine receptors and perforin accumulation, leading to enhanced migration ability and antitumor activity. Chem_NK display potent therapeutic efficacy against in vivo mouse model of triple negative breast and ovarian cancers. Fluorination of the primary amine group reduces the activity of 25KbPEI to prime NK cells, indicating that the cationic charge on the chemical plays a critical role in NK cell activation. A major priming mechanism was 25KbPEI-mediated calcium influx into NK cells, which occurred mainly via the Ca2+-permeable non-selective cation channel transient receptor potential melastatin 2. CONCLUSIONS: NK cells can be chemically primed with 25KbPEI to express potent antitumor activity as well as enhanced migration ability. Because PEI is a biocompatible and Food and Drug Administration-approved chemical for biomedical use, these results suggest a cost-effective and simple method of producing therapeutic NK cells.

ELK3 modulates the antitumor efficacy of natural killer cells against triple negative breast cancer by regulating mitochondrial dynamics.

BACKGROUND: Triple negative breast cancer (TNBC) is the most lethal subtype of breast cancer due to its aggressive behavior and frequent development of resistance to chemotherapy. Although natural killer (NK) cell-based immunotherapy is a promising strategy for overcoming barriers to cancer treatment, the therapeutic efficacy of NK cells against TNBC is below expectations. E26 transformation-specific transcription factor ELK3 (ELK3) is highly expressed in TNBCs and functions as a master regulator of the epithelial-mesenchymal transition. METHODS: Two representative human TNBC cell lines, MDA-MB231 and Hs578T, were exposed to ELK3-targeting shRNA or an ELK3-expressing plasmid to modulate ELK3 expression. The downstream target genes of ELK3 were identified using a combined approach comprising gene expression profiling and molecular analysis. The role of ELK3 in determining the immunosensitivity of TNBC to NK cells was investigated in terms of mitochondrial fission-fusion transition and reactive oxygen species concentration both in vitro and in vivo. RESULTS: ELK3-dependent mitochondrial fission-fusion status was linked to the mitochondrial superoxide concentration in TNBCs and was a main determinant of NK cell-mediated immune responses. We identified mitochondrial dynamics proteins of 51 (Mid51), a major mediator of mitochondrial fission, as a direct downstream target of ELK3 in TNBCs. Also, we demonstrated that expression of ELK3 correlated inversely with that of Mid51, and that the ELK3-Mid51 axis is associated directly with the status of mitochondrial dynamics. METABRIC analysis revealed that the ELK3-Mid51 axis has a direct effect on the immune score and survival of patients with TNBC. CONCLUSIONS: Taken together, the data suggest that NK cell responses to TNBC are linked directly to ELK3 expression levels, shedding new light on strategies to improve the efficacy of NK cell-based immunotherapy of TNBC.

The Impact of Smoking and Multiple Health Behaviors on All-Cause Mortality.

Four common health risk behaviors have the greatest impact on all-cause mortality risk, but studies are needed with larger samples and the appropriate age range for cigarette smokers. We examined the impact of smoking in the context of multiple health behaviors on all-cause mortality using a nationally representative sample of adults aged 30 and older in the United States. National Health Interview Survey data from 1997 to 2005 were linked to the National Death Index with a follow-up to December 2015. The primary dependent variable was all-cause mortality, and the primary predictors were smoking, heavy drinking, physical inactivity, and unhealthy weight (underweight or obesity). The sample contained 189,087 individuals (≥ age 30; population estimate = 140.7 million). Our primary statistical analysis tool involved fitting Cox proportional hazards models. Our findings demonstrated that smoking led to the highest mortality risk among the four risk behaviors examined, but more than half of smokers engaged in at least one additional health risk behavior. Smokers who engaged in multiple health behaviors experienced higher increased mortality risks: smoking combined with one other health risk behavior increased mortality risk by 32% and by 82% when combined with two behaviors. Engaging in all four risk behaviors more than doubled the mortality risk of smokers. Smoking cessation interventions that address multiple risk behaviors-physical inactivity, heavy drinking, and unhealthy weight-will likely prevent premature death better than interventions that address only smoking.

Constitutive Activating Eel Luteinizing Hormone Receptors Induce Constitutively Signal Transduction and Inactivating Mutants Impair Biological Activity.

In contrast to the human lutropin receptor (hLHR) and rat LHR (rLHR), very few naturally occurring mutants in other mammalian species have been identified. The present study aimed to delineate the mechanism of signal transduction by three constitutively activating mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of the eel LHR, known to be naturally occurring in human LHR transmembrane domains. The mutants were constructed and measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO)-K1 cells. The activating mutant cells expressing eel LHR-M410T, L469R, and D590Y exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response without agonist treatment, respectively. However, inactivating mutant cells expressing D417N and Y558F did not completely impaired signal transduction. Specifically, signal transduction in the cells expressing activating mutant L469R was not occurred with a further ligand stimulation, showing that the maximal response exhibited approximately 53% of those of wild type receptor. Our results suggested that the constitutively activating mutants of the eel LHR consistently occurred without agonist treatment. These results provide important information of LHR function in fish and regulation with regard to mutations of highly conserved amino acids in glycoprotein hormone receptors.

Constitutively Activating Mutants of Equine LH/CGR Constitutively Induce Signal Transduction and Inactivating Mutations Impair Biological Activity and Cell-Surface Receptor Loss In Vitro.

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts.

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.

Combined use of cisplatin plus natural killer cells overcomes immunoresistance of cisplatin resistant ovarian cancer.

Standard chemotherapy for ovarian cancers is often abrogated by drug resistance. Specifically, resistance to cisplatin is a major clinical obstacle to successful treatment of ovarian cancers. The aim of this study was to develop a therapeutic strategy using natural killer (NK) cells to treat cisplatin-resistant ovarian cancers. First, we compared the responses of ovarian cancer cell line A2780 and its cisplatin-resistant counterpart, A2780cis, to treatment with cisplatin plus NK92MI cells. Although combined treatment induces apoptosis of ovarian cancer cells via ROS-dependent and -independent mechanisms, A2780cis were resistant to NK92MI cell-mediated cytotoxicity. We found that A2780cis cells showed markedly higher expression of immune checkpoint protein, PD-L1, than the parental cells. Although pretreatment of A2780cis cells with cisplatin stimulated further expression of PD-L1, it also increased expression of ULBP ligands, which are activating receptors on NK92MI cells, both in vitro and in vivo. These findings suggest that combined use of cisplatin plus NK cell-mediated immunotherapy could overcome immunoresistance of chemoresistant ovarian cancers.