Temporal Release of Cell Cycle Regulator α-Lipoic Acid: An NIR-Light (Two-Photon) Activatable Quinoxaline-Based Nano-Prodrug Delivery System.

The regulation of the cell cycle has recently opened up a new research perspective for cancer treatment. So far, no effort has been made for temporal control of cell cycles using a photocleavable linker. Presented herein is the first report of regulation of disrupted cell cycles through the temporal release of a well-known cell cycle regulator α-lipoic acid (ALA), enabled by a newly designed NIR-active quinoxaline-based photoremovable protecting group (PRPG). The suitable quinoxaline-based photocage of ALA (tetraphenylethelene conjugated) has been formulated as fluorescent organic nanoparticles (FONs) and used effectively as a nano-DDS (drug delivery system) for better solubility and cellular internalization. Fascinatingly, the enhanced TP (two-photon) absorption cross section of the nano-DDS (503 GM) signifies its utility for biological applications. Using green light, we have successfully controlled the time span of cell cycles and cell growth of skin melanoma cell lines (B16F10) by the temporal release of ALA. Further, in silico studies and PDH activity assay supported the observed regulatory behavior of our nano-DDS with respect to photoirradiation. Overall, this approach expands the research path toward a futuristic photocontrolled toolbox for cell cycle regulation.

Fluid shear stress influences invasiveness of HeLa cells through the induction of autophagy.

Extravasation of metastatic cells from the blood or lymphatic circulation and formation of secondary tumor at a distant site is a key step of cancer metastasis. In this study, we report the role of hemodynamic shear stresses in fostering the release of pro-extravasation factors through the mediation of autophagy in cervical cancer HeLa cells. HeLa cells were exposed to physiological shear stress through the microfluidic approach adapted in our previous study on the role of hemodynamic shear stresses in survival of HeLa cells. Herein, an optimum number of passes through a cylindrical microchannel was chosen such that the viability of cells was unaffected by shear. Shear-exposed cells were then probed for their invasive and migratory potential through in vitro migration and invasion assays. The dependence of cancer cells on mechanically-induced autophagy for extravasation was further assessed through protein expression studies. Our results suggest that shear stress upregulates autophagy, which fosters paxillin turnover thereby leading to enhanced focal adhesion disassembly and in turn enhanced cell migration. Concurrently, shear stress-induced secretion of pro-invasive factors like MMP-2 and IL-6 were found to be autophagy-dependent thereby hinting at autophagy as a potential therapeutic target in metastatic cancer. Proposed model for mechano-autophagic modulation of extravasation.

Organoids: a novel modality in disease modeling.

Limitations of monolayer culture conditions have motivated scientists to explore new models that can recapitulate the architecture and function of human organs more accurately. Recent advances in the improvement of protocols have resulted in establishing three-dimensional (3D) organ-like architectures called 'organoids' that can display the characteristics of their corresponding real organs, including morphological features, functional activities, and personalized responses to specific pathogens. We discuss different organoid-based 3D models herein, which are classified based on their original germinal layer. Studies of organoids simulating the complexity of real tissues could provide novel platforms and opportunities for generating practical knowledge along with preclinical studies, including drug screening, toxicology, and molecular pathophysiology of diseases. This paper also outlines the key challenges, advantages, and prospects of current organoid systems.

Impact of seed-transmitted endophytic bacteria on intra- and inter-cultivar plant growth promotion modulated by certain sets of metabolites in rice crop.

Exploring the beneficial interactions between plant and endophytes could be an effective strategy in the implementation of sustainable agricultural practices to enhance crop productivity. In this study, we aimed to evaluate holistically the plant growth promoting (PGP) abilities rendered by seed-transmitted endophytic bacteria isolated from in vitro grown calli of two rice cultivars. Nine bacterial endophytes, designated as PB001-PB009, were isolated and identified at the genus level through 16S rRNA gene sequence analysis. Biochemical investigations disclosed that they possess several PGP traits, such as phosphate solubilization, indole acetic acid biosynthesis, ammonia production, nitrogen fixation, amylase production and siderophore production. Results in gnotobiotic conditions revealed an increase in fresh weight, dry weight, root length and shoot length of seedlings germinated from endophyte-primed seeds than the control (uninoculated) set in a non-host and two host rice cultivars. In net house experiments, plants germinated from Micrococcus sp. PB001, Pseudomonas sp. PB002, Methylobacterium sp. PB005 and Methylorubrum sp. PB009 primed seeds showed an increase of upto 34.06 %, 38.77 %, 182.87 %, 16.59 % and 33.52 % in chlorophyll content, number of tillers/plant, number of grains/plant, grain size and grain weight, respectively than control plant sets in the non-host rice cultivar, further validating inter-cultivar PGP abilities of these endophytes. Metabolite profiling unfolded the abundance of few metabolites that are involved in pathways associated with PGP traits, in seedlings germinated from the endophyte-primed seeds. Together, the study documents the effect of seed-transmitted endophytic bacteria on intra- and inter-cultivar PGP by modulating certain sets of metabolites in rice plant, and is promising in developing bioinoculant formulations employing these selected endophytes for enhancement of rice productivity.

Silanization improves biocompatibility of graphene oxide.

Evaluation of the biological properties of silanized graphene oxide is important in the context of biomedical applications of the material. In this study, we have evaluated the toxicity, immunogenicity and other biological properties like osteogenicity of silanized graphene oxide (SiGO). Graphene oxide (GO) was silanized using a common silanizing agent namely (3-aminopropyl) triethoxysilane (APTES). Silanization was confirmed through infrared spectroscopy and elemental mapping. Post-silanization, we did not observe any significant changes in the morphology of GO. Silanization leads to an increase in the interlayer distance and disorder in the lattice. Study of in vitro toxicity of SiGO on three different cell lines namely primary human dermal fibroblast, murine embryonic fibroblast and human osteosarcoma cell lines revealed that toxicity of SiGO was significantly less than GO. We further showed that in vitro immune activation of macrophage was less in the case of SiGO in comparison to GO. Profiling of osteogenic differentiation of human mesenchymal stem cell revealed that SiGO is less osteogenic than GO. Study of acute toxicity in the murine model indicated that GO was hepatotoxic at experimental concentration whereas SiGO did not show any significant toxicity. This study implied that SiGO is a better biocompatible material than GO.

Deacetylation of LAMP1 drives lipophagy-dependent generation of free fatty acids by Abrus agglutinin to promote senescence in prostate cancer.

Therapy-induced senescence in cancer cells is an irreversible antiproliferative state, which inhibits tumor growth and is therefore a potent anti-neoplastic mechanism. In this study, low doses of Abrus agglutinin (AGG)-induced senescence through autophagy in prostate carcinoma cells (PC3) and inhibited proliferation. The inhibition of autophagy with 3-methyl adenine reversed AGG-induced senescence, thus confirming that AGG-triggered senescence required autophagy. AGG treatment also led to lipophagy-mediated accumulation of free fatty acids (FFAs), with a concomitant decrease in the number of lipid droplets. Lalistat, a lysosomal acid lipase inhibitor, abrogated AGG-induced lipophagy and senescence in PC3 cells, indicating that lipophagy is essential for AGG-induced senescence. The accumulation of FFAs increased reactive oxygen species generation, a known facilitator of senescence, which was also reduced in the presence of lalistat. Furthermore, AGG upregulated silent mating type information regulator 2 homolog 1 (SIRT1), while the presence of sirtinol reduced autophagy flux and the senescent phenotype in the AGG-treated cells. Mechanistically, AGG-induced cytoplasmic SIRT1 deacetylated a Lys residue on the cytoplasmic domain of lysosome-associated membrane protein 1 (LAMP1), an autolysosomal protein, resulting in lipophagy and senescence. Taken together, our findings demonstrate a novel SIRT1/LAMP1/lipophagy axis mediating AGG-induced senescence in prostate cancer cells.

Mechanical stress-induced autophagic response: A cancer-enabling characteristic?

Metastasis is the leading cause of cancer mortality. Throughout the cascade of metastasis, cancer cells are exposed to both chemical and mechanical cues which influence their migratory behavior and survival. Mechanical forces in the milieu of cancer may arise due to excessive growth of cells in a confinement as in case of solid tumors, interstitial flows within tumors and due to blood flow in the vasculature as in case of circulating tumor cells. The focus of this review is to highlight the mechanical forces prevalent in the cancer microenvironment and discuss the impact of mechanical stresses on cancer progression, with special focus on mechanically induced autophagic response in cancer cells. Autophagy is a cellular homeostatic mechanism that a cell employs not only for recycling of damaged organelles and turnover of proteins involved in cellular migration but also as an adaptive response to survive through unfavourable stresses. Elucidation of the role of mechanically triggered autophagic response may lead to a better understanding of the mechanobiological aspects of metastatic cancer and unravelling the associated signaling mechanochemical pathways may hint at potential therapeutic targets.

Abrus agglutinin inhibits oral carcinogenesis through inactivation of NRF2 signaling pathway.

Abrus agglutinin (AGG), a heterotetrameric type II ribosome inactivating protein isolated from the seeds of Abrus precatorius shows potent antitumor activity in different cancer models. We examined the role of antioxidant system in modulation of the anticancer activity of AGG in in vitro and in hamster model of oral cancer. AGG promotes apoptosis through accumulation of ROS in CAL33 cells. Interestingly, our data showed that AGG decreases the activity of antioxidant enzymes including superoxide dismutase, catalase, glutathione peroxidase in CAL33 cells indicating antioxidant enzyme inhibition leads to AGG-induced ROS accumulation. Moreover, AGG inhibits expression of NRF2, transcription factor which regulates the expression of antioxidant enzymes in CAL33 cells. We found that AGG induces autophagy stimulation and loss of p62 expression in CAL33 cells. Furthermore, it showed that NRF2 expression is restored in the presence of 3-methyladenine and Baficomycin-A1 establishing role of autophagy in modulation of NRF2 through p62. Our study showed that AGG significantly inhibited tumor growth in DMBA-induced carcinogenesis. In immunohistochemical analysis, AGG-treated tumor displays higher caspase 3 expression and less p62 and NRF2 expression in comparison to the control. In conclusion, AGG-induced degradation of NRF2 through autophagy leads to ROS accumulation dependent apoptosis which might be used for treatment of oral cancer.

One- and Two-Photon-Activated Cysteine Persulfide Donors for Biological Targeting.

Persulfides have been considered as potential signaling compounds similar to the H2S in "S-persulfidation", a sulfur-mediated redox cycle. The research of this sulfur-mediated species is hindered because of the lack of efficient persulfide donors. In this current study, we have developed one- and two-photon-activated persulfide donors based on an o-nitrobenzyl (ONB) phototrigger, which releases the biologically active persulfide (N-acetyl l-cysteine persulfide, NAC-SSH) in a spatiotemporal manner. Next, we have demonstrated the detection of persulfide release both qualitatively and quantitatively using the well-known "turn on" fluorescence probe, that is, monobromobimane, and the trapping agent, that is, 2,4-dinitrofluorobenzene, respectively. Furthermore, we examined the cytotoxicity of synthesized persulfide donors on HeLa cells and the cytoprotective ability in the highly oxidizing cellular environment.

Role of Escherichia coli endopeptidases and dd-carboxypeptidases in infection and regulation of innate immune response.

The low-molecular-mass penicillin-binding proteins, involved in peptidoglycan recycling can also produce peptidoglycan fragments capable of activating an innate immune response in host. To investigate how these proteins in Enterobacteriaceae play a role to elicit/evade innate immune responses during infections, we deleted certain endopeptidases and dd-carboxypeptidases from Escherichia coli CS109 and studied the viability of these mutants in macrophages. The ability of infected macrophages to exert oxidative killing, express surface activation markers TLR2, MHC class II and release TNFα, were assessed. Immune responses were elevated in macrophages infected with dd-carboxypeptidase mutants but reduced for endopeptidase mutants. However, the NFκB, iNOS, and TLR2 transcripts remained elevated in macrophages infected with both mutant types. Overall, we have shown, under normal conditions endopeptidases have a tendency to elicit the immune response but their effect is suppressed by the presence of dd-carboxypeptidases. Conversely, DD-carboxypeptidases, normally, tend to reduce immune responses, as their deletions enhanced the same in macrophages. Therefore, we conclude that the roles of endopeptidases and dd-carboxypeptidases are possibly counter-active in wild-type cells where either class of enzymes suppresses each other's immunogenic properties rendering overall maintenance of low immunogenicity that helps E. coli in evading the host immune responses.

Compressive stress-induced autophagy promotes invasion of HeLa cells by facilitating protein turnover in vitro.

Metastasis remains the primary cause of cancer mortality. Throughout the process of metastasis, cancer cells experience mechanical forces, which may turn out to be the key towards their migratory, homeostatic and survival characteristics. However, the influence of compressive stress on the underlying mechanism of cancer cell adaptation during metastasis has remained grossly unexplored. In this study, we have investigated whether compressive force induces autophagy in HeLa cells with potential implications in cellular invasiveness. To this end, we have adopted a simple strategy to create the mechanically-compressed tumor microenvironment, in vitro, by applying appropriate compression to agarose-scaffolded HeLa cell-encapsulated alginate beads. Our findings confirm that compression upregulates autophagy, which promotes paxillin turnover and active MMP-2 secretion, leading to enhanced migration of HeLa cells. We further show that autophagy induction by compression is affected by the phosphorylation of p38 MAPKs, a process that is mediated by intact membrane lipid rafts. Identifying the role of such mechanically triggered cellular responses, guiding crucial processes like cell migration, may lead to better understanding of the mechanobiological aspects of metastatic cancer and unveil potential therapeutic targets.

Plant lectins in cancer therapeutics: Targeting apoptosis and autophagy-dependent cell death.

Plant lectins are non-immunoglobin in nature and bind to the carbohydrate moiety of the glycoconjugates without altering any of the recognized glycosyl ligands. Plant lectins have found applications as cancer biomarkers for recognizing the malignant tumor cells for the diagnosis and prognosis of cancer. Interestingly, plant lectins contribute to inducing cell death through autophagy and apoptosis, indicating their potential implication in cancer inhibitory mechanism. In the present review, anticancer activities of major plant lectins have been documented, with a detailed focus on the signaling circuit for the possible molecular targeted cancer therapy. In this context, several lectins have exhibited preclinical and clinical significance, driving toward therapeutic potential in cancer treatment. Moreover, several plant lectins induce immunomodulatory activities, and therefore, novel strategies have been established from preclinical and clinical investigations for the development of combinatorial treatment consisting of immunotherapy along with other anticancer therapies. Although the application of plant lectins in cancer is still in very preliminary stage, advanced high-throughput technology could pave the way for the development of lectin-based complimentary medicine for cancer treatment.

p73 induction by Abrus agglutinin facilitates Snail ubiquitination to inhibit epithelial to mesenchymal transition in oral cancer.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT), a key step in oral cancer progression, is associated with invasion, metastasis, and therapy resistance, thus targeting the EMT represents a critical therapeutic strategy for the treatment of oral cancer metastasis. Our previous study showed that Abrus agglutinin (AGG), a plant lectin, induces both intrinsic and extrinsic apoptosis to activate the tumor inhibitory mechanism. OBJECTIVE: This study aimed to investigate the role of AGG in modulating invasiveness and stemness through EMT inhibition for the development of antineoplastic agents against oral cancer. METHODS: The EMT- and stemness-related proteins were studied in oral cancer cells using Western blot analysis and fluorescence microscopy. The potential mechanisms of Snail downregulation through p73 activation in FaDu cells were evaluated using Western blot analysis, immunoprecipitation, confocal microscopy, and molecular docking analysis. Immunohistochemical staining of the tumor samples of AGG-treated FaDu-xenografted nude mice was performed. RESULTS: At the molecular level, AGG-induced p73 suppressed Snail expression, leading to EMT inhibition in FaDu cells. Notably, AGG promoted the translocation of Snail from the nucleus to the cytoplasm in FaDu cells and triggered its degradation through ubiquitination. In this setting, AGG inhibited the interaction between Snail and p73 in FaDu cells, resulting in p73 activation and EMT inhibition. Moreover, in epidermal growth factor (EGF)-stimulated FaDu cells, AGG abolished the upregulation of extracellular signal-regulated kinase (ERK)1/2 that plays a pivotal role in the upregulation of Snail to regulate the EMT phenotypes. In immunohistochemistry analysis, FaDu xenografts from AGG-treated mice showed decreased expression of Snail, SOX2, and vimentin and increased expression of p73 and E-cadherin compared with the control group, confirming EMT inhibition as part of its anticancer efficacy against oral cancer. CONCLUSION: In summary, AGG stimulates p73 in restricting EGF-induced EMT, invasiveness, and stemness by inhibiting the ERK/Snail pathway to facilitate the development of alternative therapeutics for oral cancer.

Hemodynamic shear stress induces protective autophagy in HeLa cells through lipid raft-mediated mechanotransduction.

During metastatic dissemination, cancer cells experience shear stresses in narrow confinements of in vivo vasculature. Such stresses are currently known to influence a gamut of cellular processes. While a host of cells emanating from a primary tumor perish in circulation due to shear, some cells manage to migrate to distant niches and form secondary tumors. Current research focuses on how cancer cells avert such mechanical stresses and adapt themselves in order to survive. This study deals with the autophagic response of cervical cancer cells HeLa and its subline HeLa 229, exposed to physiological shear stresses in vitro, and evaluates its role as a pro-survival mechanism. It also delineates the probable mechanotransduction pathway that is involved in eliciting the stress-adaptive response in cervical cancer cells. Our results show that shear stress of physiological regime elicits protective autophagy in cervical cancer cells as an immediate response and inhibiting the same, leads to early onset of apoptosis. An effort to study the underlying mechanotransduction revealed that autophagy induction by shear stress requires intact lipid rafts which serve as signalling platforms to trigger phosphorylation of p38 mitogen activated protein kinases, leading to autophagy. This study thus gives novel insights into the mechanobiology of cervical cancer and hints at promising therapeutic targets in metastasis, the major cause of cancer mortality.

Light triggered uncaging of hydrogen sulfide (H2S) with real-time monitoring.

An ESIPT based light activated hydrogen sulfide (H2S) donor using a p-hydroxyphenacyl phototrigger has been developed. The unique feature of our H2S donor system is that it provides real-time monitoring of H2S release by a non-invasive fluorescence colour change approach.

Abrus agglutinin stimulates BMP-2-dependent differentiation through autophagic degradation of ß-catenin in colon cancer stem cells.

Eradicating cancer stem cells (CSCs) in colorectal cancer (CRC) through differentiation therapy is a promising approach for cancer treatment. Our retrospective tumor-specimen analysis elucidated alteration in the expression of bone morphogenetic protein 2 (BMP-2) and ß-catenin during the colon cancer progression, indicating that their possible intervention through "forced differentiation" in colon cancer remission. We reveal that Abrus agglutinin (AGG) induces the colon CSCs differentiation, and enhances sensitivity to the anticancer therapeutics. The low dose AGG (max. dose = 100 ng/mL) decreased the expression of stemness-associated molecules such as CD44 and ß-catenin in the HT-29 cell derived colonospheres. Further, AGG augmented colonosphere differentiation, as demonstrated by the enhanced CK20/CK7 expression ratio and induced alkaline phosphatase activity. Interestingly, the AGG-induced expression of BMP-2 and the AGG-induced differentiation were demonstrated to be critically dependent on BMP-2 in the colonospheres. Similarly, autophagy-induction by AGG was associated with colonosphere differentiation and the gene silencing of BMP-2 led to the reduced accumulation of LC3-II, suggesting that AGG-induced autophagy is dependent on BMP-2. Furthermore, hVps34 binds strongly to BMP-2, indicating a possible association of BMP-2 with the process of autophagy. Moreover, the reduction in the self-renewal capacity of the colonospheres was associated with AGG-augmented autophagic degradation of ß-catenin through an interaction with the autophagy adaptor protein p62. In the subcutaneous HT-29 xenograft model, AGG profoundly inhibited the growth of tumors through an increase in BMP-2 expression and LC3-II puncta, and a decrease in ß-catenin expression, confirming the antitumor potential of AGG through induction of differentiation in colorectal cancer.