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Context: Multiwalled carbon nanotubes (MWCNTs) functionalized with lysine via 1,3-dipolar cycloaddition and conjugated to galactose or mannose are potential nanocarriers that can effectively bind to the lectin receptor in MDA-MB-231 or MCF-7 breast cancer cells. In this work, a method based on molecular dynamics (MD) simulation was used to predict the interaction of these functionalized MWCNTs with doxorubicin and obtain structural evidence that allows a better understanding of the drug loading and release process. The MD simulations showed that while doxorubicin only interacted with pristine MWCNTs through π-π stacking interactions, functionalized MWCNTs were also able to establish hydrogen bonds, suggesting that the functionalized groups improve doxorubicin loading. Moreover, the elevated adsorption levels observed for functionalized nanotubes further support this enhancement in loading efficiency. MD simulations also shed light on the intratumoral pH-specific release of doxorubicin from functionalized MWCNTs, which is induced by protonation of the daunosamine moiety. The simulations show that this change in protonation leads to a lower absorption of doxorubicin to the MWCNTs. The MD studies were then experimentally validated, where functionalized MWCNTs showed improved dispersion in aqueous medium compared to pristine MWCNTs and, in agreement with the computational predictions, increased drug loading capacity. Doxorubicin-loaded functionalized MWCNTs demonstrated specific release of doxorubicin in tumor microenvironment (pH = 5.0) with negligible release in the physiological pH (pH = 7.4). Furthermore, doxorubicin-free MWNCT nanoformulations exhibited insignificant cytotoxicity. The experimental studies yielded nearly identical results to the MD studies, underlining the usefulness of the method. Our functionalized MWCNTs represent promising non-toxic nanoplatforms with enhanced aqueous dispersibility and the potential for conjugation with ligands for targeted delivery of anti-cancer drugs to breast cancer cells. Methods: The computational model of a pristine carbon nanotube was created with the buildCstruct 1.2 Python script. The lysinated functionalized groups were added with PyMOL and VMD. The carbon nanotubes and doxorubicin molecules were parameterized using the general AMBER force field, and RESP charges were determined using Gaussian 09. Molecular dynamics simulations were carried out with the AMBER 20 software package. Adsorption levels were calculated using the water-shell function of cpptraj. Cytotoxicity was evaluated via a MTT assay using MDA-MB-231 and MCF-7 breast cancer cells. Drug uptake of doxorubicin and doxorubicin-loaded MWCNTs was measured by fluorescence microscopy. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) are mainly responsible for tumorigenesis, chemoresistance, and cancer recurrence. CSCs growth and progression are regulated by multiple signaling cascades including Wnt/β-catenin and Hh/GLI-1, which acts independently or via crosstalk. Targeting the crosstalk of signaling pathways would be an effective approach to control the CSC population. Both Wnt/β-catenin and Hh/GLI-1 signaling cascades are known to be regulated by p53/p21-dependent mechanism. However, it is interesting to delineate whether p21 can induce apoptosis in a p53-independent manner. Therefore, utilizing various subtypes of oral CSCs (SCC9-PEMT p53+/+p21+/+, SCC9-PEMT p53−/−p21+/+, SCC9-PEMT p53+/+p21−/− and SCC9-PEMT p53−/−p21−/−), we have examined the distinct roles of p53 and p21 in Resveratrol nanoparticle (Res-Nano)-mediated apoptosis. It is interesting to see that, besides the p53/p21-mediated mechanism, Res-Nano exposure also significantly induced apoptosis in oral CSCs through a p53-independent activation of p21. Additionally, Res-Nano-induced p21-activation deregulated the β-catenin-GLI-1 complex and consequently reduced the TCF/LEF and GLI-1 reporter activities. In agreement with in vitro data, similar experimental results were obtained in in vivo mice xenograft model. Schematic representation of Res-Nano deregulating the crosstalk of β-catenin-GLI-1 through activation of p21. (I) Plausible mechanism of β-catenin-GLI-1 interaction before Res-Nano treatment. (1a&1b) Co-localization of Wnt/β-catenin and Hh/GLI-1 pathways proteins along with p21. (2) β-catenin and GLI-1 release from their negative regulators like GSK3β, CK1α, AXIN, APC, and SUFU. (3) Formation of β-catenin-GLI-1 complex. (4) Nuclear localization of β-catenin-GLI-1 complex. (5) β-catenin and GLI-1 bind to their respective promoter binding region. (6) Activation of transcriptional target genes. (7) Cell growth and proliferation. (II) Plausible mechanism of β-catenin-GLI-1 interaction in the regulation of apoptosis after Res-Nano treatment. (1a&1b) Co-localization of Wnt/β-catenin and Hh/GLI-1 pathways proteins along with p21. (2) Res-Nano activates p21. (3) Activated p21 deregulates β-catenin-GLI-1 complex. (4) β-catenin and GLI-1 degraded in cytoplasm. (5) β-catenin and GLI-1 are unable to enter into the nucleus. (6) Downregulation of target genes. (7) Induction of apoptosis. [ABSTRACT FROM AUTHOR]

Recently, we reported that a combination of a natural, bioactive compound Resveratrol (RES) and a PARP inhibitor Olaparib (OLA) deregulated the homologous recombination (HR) pathway, and enhanced apoptosis in BRCA1-wild-type, HR-proficient breast cancer cells. Upon DNA damage, chromatin relaxation takes place, which allows the DNA repair proteins to access the DNA lesion. But whether chromatin remodeling has any role in RES + OLA-mediated HR inhibition is not known. By using in vitro and ex vivo model systems of breast cancer, we have investigated whether RES + OLA inhibits chromatin relaxation and thereby blocks the HR pathway. It was found that RES + OLA inhibited PARP1 activity, terminated PARP1-BRCA1 interaction, and deregulated the HR pathway only in the chromatin fraction of MCF-7 cells. DR-GFP reporter plasmid-based HR assay demonstrated marked reduction in HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. RES + OLA efficiently trapped PARP1 at the DNA damage site in the chromatin of MCF-7 cells. Unaltered expressions of HR proteins were found in the chromatin of PARP1-silenced MCF-7 cells, which confirmed that RES + OLA-mediated DNA damage response was PARP1-dependent. Histone Acetyltransferase (HAT) activity and histone H4 acetylation assays showed reduction in HAT activity and H4 acetylation in RES + OLA-treated chromatin fraction of cells. Western blot analysis showed that the HAT enzyme TIP60, P400 and acetylated H4 were downregulated after RES + OLA exposure. In the co-immunoprecipitation assay, it was observed that RES + OLA caused abolition of PARP1-TIP60-BRCA1 interaction, which suggested the PARP1-dependent TIP60-BRCA1 association. Unaltered expressions of PAR, BRCA1, P400, and acetylated H4 in the chromatin of TIP60-silenced MCF-7 cells further confirmed the role of TIP60 in PARP1-mediated HR activation in the chromatin. Similar results were obtained in ex vivo patient-derived primary breast cancer cells. Thus, the present study revealed that RES + OLA treatment inhibited PARP1 activity in the chromatin, and blocked TIP60-mediated chromatin relaxation, which, in turn, affected PARP1-dependent TIP60-BRCA1 association, resulting in deregulation of HR pathway in breast cancer cells. [ABSTRACT FROM AUTHOR]

The presence of cancer stem cells (CSCs) in the tumor microenvironment (TME) is majorly responsible for the development and recurrence of cancer. Earlier reports suggested that upon DNA damage, poly-(ADP-ribose) polymerase-1 (PARP-1) helps in chromatin modulation and DNA repair process, thereby promoting CSC survival. But whether a combination of DNA damaging agents along with PARP inhibitors can modulate chromatin assembly, inhibit DNA repair processes, and subsequently target CSCs is not known. Hence, we have investigated the effect of nontoxic bioactive compound quinacrine (QC) and a potent PARP inhibitor Talazoparib in patient-derived oral mucosa CSCs (OM-CSCs) and in vivo xenograft mice preclinical model systems. Data showed that QC + Talazoparib inhibited the PARP-1-mediated chromatin remodelers' recruitment and deregulated HAT activity of GCN5 (general control nonderepressible-5) and P300 at DNA damage site, thereby preventing the access of repair proteins to the damaged DNA. Additionally, this combination treatment inhibited topoisomerase activity, induced topological stress, and induced apoptosis in OM-CSCs. Similar results were observed in an in vivo xenograft mice model system. Collectively, the data suggested that QC + Talazoparib treatment inhibited BER pathway, induced genomic instability and triggered apoptosis in OM-CSCs through the deregulation of PARP-1-mediated chromatin remodelers (GCN5 and P300) activity. Schematic representation of QC + Talazoparib-induced apoptosis in oral mucosa CSCs. (1) Induction of DNA damage takes place after QC treatment (2) PARP1-mediated PARylation at the site of DNA damage, which recruits multiple chromatin remodelers (3) Acetylation at the histone tails relax the structure of chromatin and recruits the BER pathway proteins at the site of DNA damage. (4) BER pathway activated at the site of DNA damage. (5) CSCs survive after successful repair of DNA damage. (6) Treatment of QC-treated CSCs with PARP inhibitor Talazoparib (7) Inhibition of PARylation results in failure of chromatin remodelers to interact with PARP1. (8) Inhibition of acetylation status leads to chromatin compaction. (9) BER pathway proteins are not recruited at the site of DNA damage, resulting in inhibition of BER pathway and accumulation of unrepaired DNA damage, leading to apoptosis and cell death. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) cause drug resistance in cancer due to its extensive drug efflux, DNA repair and self-renewal capability. ATP binding cassette subfamily G member 2 (ABCG2) efflux pump afford protection to CSCs in tumors, shielding them from the adverse effects of chemotherapy. Although the role of ABCG2 in cancer progression, invasiveness, recurrence are known but its role in metastasis and angiogenesis are not clear. Here, using in vitro (CSCs enriched side population [SP] cells), ex vivo (patient derived primary cells), in ovo (fertilized egg embryo) and in vivo (patient derived primary tissue mediated xenograft (PDX)) system, we have systematically studied the role of ABCG2 in angiogenesis and the regulation of the process by Curcumin (Cur) and Quinacrine (QC). Cur + QC inhibited the proliferation, invasion, migration and expression of representative markers of metastasis and angiogenesis. Following hypoxia, ABCG2 enriched cells released angiogenic factor vascular endothelial growth factor A (VEGF A) and induced the angiogenesis via PI3K-Akt-eNOS cascade. Cur + QC inhibited the ABCG2 expression and thus reduced the angiogenesis. Interestingly, overexpression of ABCG2 in SP cells and incubation of purified ABCG2 protein in media induced the angiogenesis but knockdown of ABCG2 decreased the vascularization. In agreement with in vitro results, ex vivo data showed similar phenomena. An induction of vascularization was noticed in PDX mice but reduction of vascularization was also observed after treatment of Cur + QC. Thus, data suggested that in hypoxia, ABCG2 enhances the production of angiogenesis factor VEGF A which in turn induced angiogenesis and Cur + QC inhibited the process by inhibiting ABCG2 in breast cancer. [ABSTRACT FROM AUTHOR]

Wnt, Hedgehog (Hh), and Notch signaling pathways are the evolutionarily conserved signaling pathways that regulate the embryonic development and also play crucial role in maintaining stemness properties of cancer stem cells (CSCs) and inducing epithelial-to-mesenchymal transition (EMT), metastasis, and angiogenesis. It has been highly challenging to inhibit the CSCs growth and proliferation as these are capable of evading chemotherapeutic drugs and cause cancer recurrence through multiple signaling pathways. Therefore, novel therapeutic strategies to target the key players involved in the crosstalk of these signaling pathways need to be developed. In this review, we have identified the interacting molecules of Wnt, Hh, and Notch pathways responsible for enhancing the malignant properties of CSCs. Analyzing the functions of these crosstalk molecules will help us to find an approach toward the development of new anti-cancer drugs for inhibition of CSCs growth and progression. Long non-coding RNAs (LncRNAs) play a significant role in various cellular processes, like chromatin remodeling, epigenetic modifications, transcriptional, and post-transcriptional regulations. Here, we have highlighted the research findings suggesting the involvement of LncRNAs in maintenance of the stemness properties of CSCs through modulation of the above-mentioned signaling pathways. We have also discussed about the different therapeutic approaches targeting those key players responsible for mediating the crosstalk between the pathways. Overall, this review article will surely help the cancer biologists to design novel anti-CSCs agents that will open up a new horizon in the field of anti-cancer therapeutics. [ABSTRACT FROM AUTHOR]