Enigmatic exosomal connection in lung cancer drug resistance.

Lung cancer remains a significant global health concern with limited treatment options and poor prognosis, particularly in advanced stages. Small extracellular vesicles such as exosomes, secreted by cancer cells, play a pivotal role in mediating drug resistance in lung cancer. Exosomes have been found to facilitate intercellular communication by transferring various biomolecules between cancer cells and their microenvironment. Additionally, exosomes can transport signaling molecules promoting cancer cell survival and proliferation conferring resistance to chemotherapy. Moreover, exosomes can modulate the tumor microenvironment by inducing phenotypic changes hindering drug response. Understanding the role of exosomes in mediating drug resistance in lung cancer is crucial for developing novel therapeutic strategies and biomarkers to overcome treatment limitations. In this review, we summarize the current knowledge on conventional and emerging drug resistance mechanisms and the involvement of exosomes as well as exosome-mediated factors mediating drug resistance in lung cancer.

Transforming native exosomes to engineered drug vehicles: A smart solution to modern cancer theranostics.

Exosomes have been the hidden treasure of the cell in terms of cellular interactions, transportation and therapy. The native exosomes (NEx) secreted by the parent cells hold promising aspects in cancer diagnosis and therapy. NEx has low immunogenicity, high biocompatibility, low toxicity and high stability which enables them to be an ideal prognostic biomarker in cancer diagnosis. However, due to heterogeneity, NEx lacks specificity and accuracy to be used as therapeutic drug delivery vehicle in cancer therapy. Transforming these NEx with their innate structure and multiple receptors to engineered exosomes (EEx) can provide better opportunities in the field of cancer theranostics. The surface of the NEx exhibits numeric receptors which can be modified to pave the direction of its therapeutic drug delivery in cancer therapy. Through surface membrane, EEx can be modified with increased drug loading potentiality and higher target specificity to act as a therapeutic nanocarrier for drug delivery. This review provides insights into promising aspects of NEx as a prognostic biomarker and drug delivery tool along with its need for the transformation to EEx in cancer theranostics. We have also highlighted different methods associated with NEx transformations, their nano-bio interaction with recipient cells and major challenges of EEx for clinical application in cancer theranostics.

Exposure of piperlongumine attenuates stemness and epithelial to mesenchymal transition phenotype with more potent anti-metastatic activity in SOX9 deficient human lung cancer cells.

Phytocompounds have shown hopeful results in cancer therapy. Piperlongumine (PIP), a naturally derived bioactive alkaloid found in our dietary spice, exhibits promising pharmacological relevance including anticancer activity. This study reconnoitred the anti-lung cancer effect of PIP and the allied mechanisms, in vitro and ex vivo. The cytotoxic, anti-proliferative, and apoptotic effects of PIP on lung cancer cells (LCC) were checked via cell viability, colony formation, cell migration, invasion, comet assay, and various staining techniques. Further, multicellular spheroids assay explored the anti-lung cancer potential of PIP, ex vivo. Preliminary results explored that PIP exerts selective cytotoxic and anti-proliferative effects on LCC by DNA damage and cell cycle arrest. PIP remarkably escalated the cellular and mitochondrial reactive oxygen species (ROS) generation and promoted dissipation of mitochondrial membrane potential (MMP), which triggers activation of caspase-dependent apoptotic pathway in LCC. Mechanistically, PIP showed F-actin deformation mediated significant anti-migratory and anti-invasive activity against LCC. Herein, we also found that F-actin dis-organization modulates the expression of epithelial to mesenchymal transition (EMT) markers and inhibits the expression of stemness marker proteins, like SOX9, CD-133, and CD-44. Moreover, PIP effectively reduced the size of spheroids with strong apoptotic and cytotoxic effects, ex vivo. This has been the first study to discover the high expression of SOX9 supporting the survival of LCC, whereas its inhibition induces higher sensitivity to PIP treatment. This study concludes a newer therapeutic agent (PIP) with promising anticancer activity against LCC by escalating ROS and attenuating MMP, stemness, and EMT.

Synthesis of First Coumarin Fluorescent Dye for Actin Visualization.

Selective fluorescence imaging of actin protein hugely depends on the fluorescently labeled actin-binding domain (ABD). Thus, it is always a challenging task to image the actin protein (in vivo or in vitro) directly with an ABD-free system. To overcome the limitations of actin imaging without an ABD, we have designed a facile and cost-effective red fluorescent coumarin dye 7-hydroxy-4-methyl-8-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl-2H-chromen-2-one (CTC) for actin binding. The selective binding of the dye was investigated using the gut and eye of the model organism Drosophila melanogaster and C2C12 and SCC-9 cell lines. Our result suggests two major advantages of CTC over the dyes presently used for imaging actin proteins. First, the dye can bind to actin efficiently without any secondary intermediate. Second, it is much more stable at room temperature and exhibits excellent photostability. To the best of our knowledge, this is the first fluorescent dye that can bind to the actin protein without employing any secondary intermediate/actin-binding domain. These findings could pave the way for many biologists and physicists to successfully employ the CTC dye for imaging and tracking actin proteins by fluorescence microscopy in various in vivo and in vitro systems.

SOX9 as an emerging target for anticancer drugs and a prognostic biomarker for cancer drug resistance.

The dysregulated expression of the transcription factor (TF) Sry-related HMG box 9 (SOX9) has been extensively correlated with various biological effects, including the initiation and progression of cancer. Differential expression of SOX9 has been positively correlated with cancer cell growth, invasion, migration, metastasis, and therapy resistance. Studies showed that expression of SOX9 affects the expression of various miRNAs and vice versa, resulting in the development of cancer drug resistance. However, modulating the expression of SOX9 reverses drug resistance by modulating the expression of miRNAs. Therefore, in this review, we summarize current research focusing on SOX9 as a cancer therapeutic target and a prognostic biomarker of cancer drug resistance.

SOX9 promotes epidermal growth factor receptor-tyrosine kinase inhibitor resistance via targeting ß-catenin and epithelial to mesenchymal transition in lung cancer.

AIMS: The first-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), gefitinib, continues to be a primary treatment option for lung cancer patients. However, acquisition of resistance to gefitinib is a major obstacle in lung cancer treatment and its cause is poorly understood. The present study aimed to implicate the role of SOX9-ß-catenin in developed resistance to gefitinib through epithelial to mesenchymal transition (EMT) in lung cancer in vitro and ex vivo. MAIN METHODS: Expression effect of SOX9 on survivability of lung cancer patients was demonstrated through online available Kaplan-Meier Plotter data base. Then, cell viability assay, colony forming assay, cell migration and invasion assays, flow cytometry, drug efflux assay, qRT-PCR, and western blotting were conducted to confirmed the role of SOX9 in gefitinib resistance in lung cancer cells. Dual-luciferase assay established the regulatory relation between SOX9 and ß-catenin. Multicellular spheroid assay further explored that down regulation of SOX9 could reverse gefitinib resistance ex vivo. KEY FINDINGS: Kaplan-Meier method correlated the higher expression of SOX9 and ß-catenin with poor overall survival of lung cancer patients. Upregulation of SOX9 was associated gefitinib resistance with increased cell proliferation, migration and invasion, single-cell colony-forming ability, reduced apoptosis, and gefitinib intake in lung cancer cells. Moreover, upregulated SOX9 promoted EMT via targeting ß-catenin and knockdown of SOX9 reversed the resistance and EMT phenotype. Similarly, we found that multicellular spheroid of gefitinib resistant cells showed larger surface area with more dispersion and viability of cells, while SOX9 knockdown abolished these induced properties ex vivo. SIGNIFICANCE: SOX9 expression could provide an innovative perspective as biomarker to understand the EGFR-TKIs resistance in lung cancer.

Electroformation of liposomes and phytosomes using copper electrode.

A novel method for electroformation of liposomes and phytosomes using copper electrode is described. Liposomes made at 2 V and 10 Hz AC field from L-α-egg-phosphatidylcholine (egg-PC), K. pneumoniae phosphatidylethanolamine, K. pneumoniae polar lipids and E. coli polar lipids on copper electrode were (777.9 ± 118.4), (370.2 ± 100.5), (825.3 ± 21.54), and (281.3 ± 42.3) nm in diameter, respectively. Giant vesicles were formed at 30 V and 10 Hz AC field from polar lipids of K. pneumoniae and E. coli were (106 ± 29.7) and (86 ± 24.3) µm in diameter, respectively. All liposomes were unilamellar as indicated by their unilamellar indices of 50 ± 2, had surface charge comparable to vesicles made from lipid(s) with similar composition and exhibited only 1-2 mol% of oxidized lipids. Cu concentration in the liposomal samples was <1.5 ppm for large unilamellar vesicles (LUVs) and ˂5 ppm for giant unilamellar vesicles (GUVs). The vesicles were stable for >15 d without loss of their size, charge, or unilamellarity. The method was successfully applied to prepare phytosomes from egg-PC and a phytochemical fraction of Dimorphocalyx glabellus, a medicinal plant with anti-diuretic properties. Phytosomes formed were 1000-1500 nm in diameter and exhibited altered fluorescence and absorbance properties compared to the unencapsulated phytochemical. Phytosomes with phytochemical: egg-PC ratio from 0.15 to 1.5 had encapsulation efficiency ranging 90-30%, respectively, and was stable for 1 month. Our method is easy, inexpensive and convenient that will prove to be useful for preparation of liposomes and phytosomes.

Piperlongumine, a potent anticancer phytotherapeutic: Perspectives on contemporary status and future possibilities as an anticancer agent.

Piperlongumine, a white to beige biologically active alkaloid/amide phytochemical, has high pharmacological relevance as an anticancer agent. Piperlongumine has several biological activities, including selective cytotoxicity against multiple cancer cells of different origins at a preclinical level. Several preclinical studies have documented the anticancer potential of piperlongumine through its targeting of multiple molecular mechanisms, such as cell cycle arrest, anti-angiogenesis, anti- invasive and anti-metastasis pathways, autophagy pathways, and intrinsic apoptotic pathways in vitro and in vivo. Mechanistically, piperlongumine inhibits cancer growth by resulting in the accumulation of intracellular reactive oxygen species, decreasing glutathione and chromosomal damage, or modulating key regulatory proteins, including PI3K, AKT, mTOR, NF-kß, STATs, and cyclin D1. Furthermore, combined treatment with piperlongumine potentiates the anticancer activity of conventional chemotherapeutics and overcomes resistance to chemo- and radio- therapy. Nanoformulation of piperlongumine has been associated with increased aqueous solubility and bioavailability and lower toxicity, thus enhancing therapeutic efficacy in both preclinical and clinical settings. The current review highlights anticancer studies on the occurrence, chemical properties, chemopreventive mechanisms, toxicity, bioavailability, and pharmaceutical relevance of piperlongumine in vitro and in vivo.

Plumbagin engenders apoptosis in lung cancer cells via caspase-9 activation and targeting mitochondrial-mediated ROS induction.

Plumbagin is a naturally-derived phytochemical which exhibits promising medicinal properties, including anticancer activities. In the present study, the anticancer potential of plumbagin has been demonstrated in lung cancer cells by targeting reactive oxygen species (ROS) and the intrinsic mitochondrial apoptotic pathway. Plumbagin showed impressive cytotoxic, anti-proliferative, and anti-migratory activities with IC50 3.10 ± 0.5 µM and 4.10 ± 0.5 µM in A549 and NCI-H522 cells, respectively. Plumbagin treatment significantly reduced the size of A549 tumor spheroids in a concentration-dependent manner. Plumbagin enhanced ROS production and arrested lung cancer cells in S and G2/M phase. Expression of antioxidant genes such as glutathione S-transferase P1 and superoxide dismutase-2 were found to be upregulated with plumbagin treatment in A549 cells. Plumbagin induced dissipation in mitochondrial membrane potential and affected the expression of intrinsic apoptotic pathway proteins. Increased expression of cytochrome c promotes the activation of pro-apoptotic protein Bax with decreased expression of anti-apoptotic protein Bcl-2. Further, plumbagin activated the mitochondrial downstream pathway protein caspase-9 and caspase-3 leading to apoptosis of A549 cells. Collectively, plumbagin could be a promising future phytotherapeutic candidate for lung cancer treatment via targeting intrinsic mitochondrial apoptotic pathway and ROS.

Metal-free semi-aromatic polyester as nanodrug carrier: A novel tumor targeting drug delivery vehicle for potential clinical application.

Polyester nanomaterials have been widely used in drug delivey application from a longer period of time. This study reports the synthesis of metal-free semi-aromatic polyester (SAP) nanomaterial for drug delivery and evaluate its in vivo acute and systemic toxicity for potential clinical application. The ring opening coplymerization of commercially available cyclohexene oxide (CHO) and phthalic anhydride (PA) monomers was carried out to synthesize fully alternating poly(CHO-co-PA) copolymer using metal-free activators. The obtained low Mn SAP was found to be biocompatible, hemocompataible and biodegradable nature. This copolymer was first-time used to fabricate curcumin (CUR) loaded nanoparticles (NPs). These NPs were physicochemically characterized by thermogravimetric analyzer (TGA), X-ray diffraction (XRD), and UV/visible spectrophotometer analysis. Further, these negatively charged core-shell spherical NPs exhibited slow sustained release behavior of CUR with anomalous transport and further displayed its higher intracellular uptake in SiHa cells at different time-periods compared to free CUR. In vitro anti-cancer therapeutic effects of free CUR and poly(CHO-alt-PA)-CUR NPs were evaluated on different cancer cells. We observed the increased cytotoxicity of CUR NPs with low IC50 values compared to free CUR. These results were further substantiated with ex vivo data where, a significant reduction was observed in CUR NPs treated tumor spheroid's size as compared to free CUR. Furthermore, the different doses of metal-free poly(CHO-alt-PA) nanomaterial were tested for its acute and systemic toxicity in BALB/c mice. We did not observe any significant toxicity of tested nanomaterial on vital organs, blood cells and the body weight of mice. Our study suggest that this metal-free SAP nanomaterial can be used for potential clinical application.

An efficient protocol for deriving liver stem cells from neonatal mice: validating its differentiation potential.

The success of liver regeneration depends on the availability of suitable cell types and their potential to differentiate into functional hepatocytes. To identify the stem cells which have the ability to differentiate into hepatocytes, we used neonatal liver as source. However, the current protocol for isolating stem cells from liver involves enzymes like collagenase, hyaluronidase exposed for longer duration which limits the success. This results in the keen interest to develop an easy single step enzyme digestion protocol for isolating stem cells from liver for tissue engineering approaches. Thus, the unlimited availability of cell type favors setting up the functional recovery of the damaged liver, ensuring ahead success towards treating liver diseases. We attempted to isolate liver stem derived cells (LDSCs) from mouse neonatal liver using single step minimal exposure to enzyme followed by in vitro culturing. The cells isolated were characterized for stem cell markers and subjected to lineage differentiation. Further, LDSCs were induced to hepatocyte differentiation and validated with hepatocyte markers. Finally, we developed a reproducible, efficient protocol for isolation of LDSCs with functional hepatocytes differentiation potential, which further can be used as in vitro model system for assessing drug toxicity assays in various preclinical trials.

Differential usage of the transport systems for folic acid and methotrexate in normal human T-lymphocytes and leukemic cells.

Methotrexate (MTX) has been used as an effective anti-cancer drug for a long time. Conceptually, it is accepted that MTX and folic acid are transported by folate receptors (FRs) in cancerous cells, but the exact mechanism of MTX uptake in human leukemia is unknown. The objective of this study was to investigate different transport systems for FA and MTX, and to delineate their uptake mechanism in MOLT4, K562, Hut78 leukemia cells and normal human T cells. In MOLT4, uptake of MTX was higher than FA, similar to that of K562, Hut78 and normal T cells. In MOLT4 cells, MTX uptake was maximum at pH 7.4 whereas FA uptake was maximum at pH 4.5. Uptake of FA and MTX was significantly inhibited by anions, suggesting anion-dependent transport system. FA uptake was found to be energy dependent whereas MTX uptake was energy independent. RT-PCR and immunofluorescence results demonstrated the presence of reduced folate carrier as well as proton coupled folate transporter and absence of FR in MOLT4 and normal T cells. These data suggest the existence of two separate and independent carrier-mediated transport systems for the uptake of FA and MTX in normal and leukemic human T cells.