Therapeutic and diagnostic applications of carbon nanotubes in cancer: recent advances and challenges.

Carbon nanotubes (CNTs) are allotropes of carbon, composed of carbon atoms forming a tube-like structure. Their high surface area, chemical stability, and rich electronic polyaromatic structure facilitate their drug-carrying capacity. Therefore, CNTs have been intensively explored for several biomedical applications, including as a potential treatment option for cancer. By incorporating smart fabrication strategies, CNTs can be designed to specifically target cancer cells. This targeted drug delivery approach not only maximizes the therapeutic utility of CNTs but also minimizes any potential side effects of free drug molecules. CNTs can also be utilised for photothermal therapy (PTT) which uses photosensitizers to generate reactive oxygen species (ROS) to kill cancer cells, and in immunotherapeutic applications. Regarding the latter, for example, CNT-based formulations can preferentially target intra-tumoural regulatory T-cells. CNTs also act as efficient antigen presenters. With their capabilities for photoacoustic, fluorescent and Raman imaging, CNTs are excellent diagnostic tools as well. Further, metallic nanoparticles, such as gold or silver nanoparticles, are combined with CNTs to create nanobiosensors to measure biological reactions. This review focuses on current knowledge about the theranostic potential of CNT, challenges associated with their large-scale production, their possible side effects and important parameters to consider when exploring their clinical usage.

The Collagen-Modifying Enzyme PLOD2 Is Induced and Required during L1-Mediated Colon Cancer Progression.

The overactivation of Wnt/ß-catenin signaling is a hallmark of colorectal cancer (CRC) development. We identified the cell adhesion molecule L1CAM (L1) as a target of ß-catenin-TCF transactivation in CRC cells. The overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis and liver metastasis, and L1 is exclusively localized in the invasive areas of human CRC tissue. A number of genes are induced after L1 transfection into CRC cells by a mechanism involving the cytoskeletal protein ezrin and the NF-κB pathway. When studying the changes in gene expression in CRC cells overexpressing L1 in which ezrin levels were suppressed by shRNA to ezrin, we discovered the collagen-modifying enzyme lysyl hydroxylase 2 (PLOD2) among these genes. We found that increased PLOD2 expression was required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis and liver metastasis, since the suppression of endogenous PLOD2 expression, or its enzymatic activity, blocked the enhanced tumorigenic properties conferred by L1. The mechanism involved in increased PLOD2 expression by L1 involves ezrin signaling and PLOD2 that affect the SMAD2/3 pathway. We found that PLOD2 is localized in the colonic crypts in the stem cell compartment of the normal mucosa and is found at increased levels in invasive areas of the tumor and, in some cases, throughout the tumor tissue. The therapeutic strategies to target PLOD2 expression might provide a useful approach for CRC treatment.

A Necessary Role for Increased Biglycan Expression during L1-Mediated Colon Cancer Progression.

Aberrant activation of Wnt/ß-catenin signaling and downstream ß-catenin-TCF target genes is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin-like cell adhesion receptor L1CAM (L1) as a target of ß-catenin-TCF transactivation in CRC cells. Overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis, and liver metastasis, and L1 is exclusively localized at invasive areas of human CRC tissue. Several genes are induced after L1 transfection into CRC cells by a mechanism involving the L1-ezrin-NF-κB pathway. We conducted a secretomic analysis of the proteins in the culture medium of L1-overexpressing CRC cells. We detected a highly increased level of biglycan, a small leucine-rich ECM component, and a signaling molecule. We found that induction of biglycan is required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis, and liver metastasis. The suppression of endogenous biglycan levels or a point mutation in the L1 ectodomain that regulates cell-cell adhesion mediated by L1 blocked the enhanced tumorigenic properties conferred by L1. The mechanism of biglycan induction by L1 involves the L1-NF-κB pathway. Blocking NF-κB signaling in L1 expressing cells suppressed the induction of biglycan and the tumorigenic properties conferred by L1. Biglycan expression was undetectable in the normal colonic mucosa, but expressed at highly increased levels in the tumor tissue, especially in the stroma. The therapeutic strategies to target biglycan expression might provide a useful approach for CRC treatment in L1-overexpressing tumors.

Wnt/ß-Catenin Target Genes in Colon Cancer Metastasis: The Special Case of L1CAM.

Cell adhesion to neighboring cells is a fundamental biological process in multicellular organisms that is required for tissue morphogenesis. A tight coordination between cell-cell adhesion, signaling, and gene expression is a characteristic feature of normal tissues. Changes, and often disruption of this coordination, are common during invasive and metastatic cancer development. The Wnt/ß-catenin signaling pathway is an excellent model for studying the role of adhesion-mediated signaling in colorectal cancer (CRC) invasion and metastasis, because ß-catenin has a dual role in the cell; it is a major adhesion linker of cadherin transmembrane receptors to the cytoskeleton and, in addition, it is also a key transducer of Wnt signaling to the nucleus, where it acts as a co-transcriptional activator of Wnt target genes. Hyperactivation of Wnt/ß-catenin signaling is a common feature in the majority of CRC patients. We found that the neural cell adhesion receptor L1CAM (L1) is a target gene of ß-catenin signaling and is induced in carcinoma cells of CRC patients, where it plays an important role in CRC metastasis. In this review, we will discuss studies on ß-catenin target genes activated during CRC development (in particular, L1), the signaling pathways affected by L1, and the role of downstream target genes activated by L1 overexpression, especially those that are also part of the intestinal stem cell gene signature. As intestinal stem cells are highly regulated by Wnt signaling and are believed to also play major roles in CRC progression, unravelling the mechanisms underlying the regulation of these genes will shed light on both normal intestinal homeostasis and the development of invasive and metastatic CRC.

Increased expression of cathepsin D is required for L1-mediated colon cancer progression.

Hyperactivation of Wnt/ß-catenin target genes is considered a key step in human colorectal cancer (CRC) development. We previously identified the immunoglobulin-like cell adhesion receptor L1 as a target gene of ß-catenin/TCF transactivation that is localized at the invasive edge of CRC tissue. Using gene arrays, we discovered a number of downstream target genes and signaling pathways conferred by L1 overexpression during colon cancer progression. Here, we have used a proteomic approach to identify proteins in the secretome of L1-overexpressing CRC cells and studied the role of the increase in the aspartate protease cathepsin D (CTSD) in L1-mediated colon cancer development. We found that in addition to the increase in CTSD in the secretome, the RNA and protein levels of CTSD were also induced by L1 in CRC cells. CTSD overexpression resulted in elevated proliferation under stress and increased motility, tumorigenesis and liver metastasis, although to a lesser extent than after L1-transfection. The suppression of endogenous CTSD in L1-expressing cells blocked the increase in the proliferative, motile, tumorigenic and metastatic ability of CRC cells. Enhancing Wnt/ß-catenin signaling by the inhibition of GSK3ß resulted in increased endogenous CTSD levels, suggesting the involvement of the Wnt/ß-catenin pathway in CTSD expression. In human CRC tissue, CTSD was detected in epithelial cells and in the stromal compartment at the more invasive areas of the tumor, but not in the normal mucosa, indicating that CTSD plays an essential role in CRC progression.

ISG15 induction is required during L1-mediated colon cancer progression and metastasis.

Hyperactivation of Wnt/ß-catenin target gene expression is a hallmark of colorectal cancer (CRC) development. We identified L1-CAM (L1) and Nr-CAM, members of the immunoglobulin family of nerve cell adhesion receptors, as target genes of the Wnt/ß-catenin pathway in CRC cells. L1 overexpression in CRC cells enhances their motile and tumorigenic capacity and promotes liver metastasis. L1 is often localized at the invasive edge of CRC tissue. Using gene arrays and proteomic analyses we identified downstream signaling pathways and targets of L1-mediated signaling. Here, we found that the expression of interferon-stimulated gene 15 (ISG15) that operates much like ubiquitin (is conjugated to proteins by ISGylation), is elevated in the conditioned medium and in CRC cells overexpressing L1. Suppression of endogenous ISG15 levels in L1-expressing cells blocked the increased proliferative, motile, tumorigenic and liver metastatic capacities of CRC cells. ISG15 overexpression, on its own, could enhance these properties in CRC cells, but only to a much lower extent compared to L1. We show that NF-κB signaling is involved in the L1-mediated increase in ISG15, since blocking the NF-κB pathway abolished the induction of ISG15 by L1. Point mutations in the L1 ectodomain that interfere with its binding to L1 ligands, also inhibited the increase in ISG15. We detected high levels of ISG15 in human CRC tissue cells and in the adjacent stroma, but not in the normal mucosa. The results suggest that ISG15 is involved in L1-mediated CRC development and is a potential target for CRC therapy.

Insights into the structure and tubulin-targeted anticancer potential of N-(3-bromobenzyl) noscapine.

BACKGROUND: Noscapine is a non-narcotic, antitussive alkaloid isolated from plants of Papaveraceae family. This benzylisoquinoline alkaloid and its synthetic derivatives, called noscapinoids, are being evaluated for their anticancer potential. METHODS: The structure of a novel analogue, N-(3-bromobenzyl) noscapine (N-BBN) was elucidated by X-ray crystallography. Effect of N-BBN on cancer cell proliferation and cellular microtubules were studied by sulphorhodamine B assay and immunofluorescence, respectively. Binding interactions of the alkaloid with tubulin was studied using spectrofluorimetry. RESULTS: N-BBN, synthesized by introducing modification at site B ('N' in isoquinoline unit) and a bromo group at the 9th position of the parent compound noscapine, was found to be superior to many of the past-generation noscapinoids in inhibiting cancer cell viability and it showed a strong inhibition of the clonogenic potential of an aggressively metastatic breast tumour cell line, MDA-MB-231. The compound perturbed the tertiary structure of purified tubulin as indicated by an anilinonaphthalene sulfonic acid-binding assay. However, substantiating the common feature of noscapinoids, it did not alter microtubule polymer mass considerably. In cells, the drug-treatment showed a peculiar type of disruption of normal microtubule architecture. CONCLUSION: N-BBN may be considered for further investigations as a potent antiproliferative agent.

Cell-cell adhesion: linking Wnt/ß-catenin signaling with partial EMT and stemness traits in tumorigenesis.

Changes in cell adhesion and motility are considered key elements in determining the development of invasive and metastatic tumors. Co-opting the epithelial-to-mesenchymal transition (EMT) process, which is known to occur during embryonic development, and the associated changes in cell adhesion properties in cancer cells are considered major routes for tumor progression. More recent in vivo studies in tumor tissues and circulating tumor cell clusters suggest a stepwise EMT process rather than an "all-or-none" transition during tumor progression. In this commentary, we addressed the molecular mechanisms underlying the changes in cell adhesion and motility and adhesion-mediated signaling and their relationships to the partial EMT states and the acquisition of stemness traits by cancer cells.

Aqueous extract of Triphala inhibits cancer cell proliferation through perturbation of microtubule assembly dynamics.

Triphala (Trl) is an ayurvedic formulation used for treating disorders of the digestive, respiratory, and nervous systems. Its anticancer properties have also been documented. We studied effects of Trl on tubulin, a target protein for several anticancer drugs, and systematically elucidated a possible antiproliferative mechanism of action of Trl. Trl inhibited proliferation of HeLa (cervical adenocarcinoma), PANC-1 (pancreatic adenocarcinoma), and MDA-MB-231 (triple-negative breast carcinoma) cells in microgram quantities and strongly suppressed the clonogenicity of HeLa cells. The formulation disrupted secondary conformation of tubulin and inhibited anilino naphthalene sulfonate binding to tubulin. In cells, Trl-tubulin interactions were manifested as a perturbed microtubule network. Acetylation pattern of Trl-treated cellular microtubules indicated persistent stabilization of microtubule dynamics. In addition, Trl interfered with reassembly of the microtubules. Cells treated with Trl eventually underwent programmed cell death as evidenced by annexin-V staining. Our study shows that the effect of aqueous extract of Trl is potent enough to interfere with the assembly dynamics of microtubules, and that Trl can be investigated further for its antitumor potential.

Safranal Inhibits HeLa Cell Viability by Perturbing the Reassembly Potential of Microtubules.

Saffron, a spice from Crocus sativus, has been known for its health benefits and medicinal properties. Safranal is a component of saffron and is known for its antioxidant and anticancer properties. In this study, we elucidated a possible tubulin-targeted antiproliferative mechanism of action of safranal. In vitro, the compound perturbed secondary structure of tubulin without altering net microtubule polymer mass. It inhibited HeLa cell viability in a concentration-dependent manner, with minimal damage to cellular microtubules. However, it strongly inhibited recovery of microtubule network after cold-induced disassembly, indicating its ability to interfere with the nucleation potential of tubulin. Further, as the acetylation pattern of the safranal-treated microtubules revealed, unlike many tubulin-targeted agents, the compound did not appear to induce persistent stabilization of microtubules. Our data shows an unusual, tubulin-targeted antiproliferative mechanism of safranal. Copyright © 2017 John Wiley & Sons, Ltd.

Tryptone-stabilized gold nanoparticles target tubulin and inhibit cell viability by inducing an unusual form of cell cycle arrest.

Gold nanoparticles have been investigated extensively for their molecular mechanisms of action and anticancer potential. We report a novel, tubulin-targeted antiproliferative mechanism of action of tryptone-stabilized gold nanoparticles (TsAuNPs). TsAuNPs, synthesized using HAuCl4·3H2O and tryptone and characterized by a variety of spectroscopic methods and transmission electron microscopy, were found to be inhibitory to viability of human pancreatic (PANC-1), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines in a concentration-dependent manner, with highest efficacy against PANC-1 cells. The particles strongly inhibited the clonogenic propagation of PANC-1 cells. TsAuNPs-mediated inhibition of cell viability involved an unusual mode of cell cycle arrest (arrest at both G0/G1 phase and S-phase) followed by apoptosis. In vitro, TsAuNPs bound purified tubulin, competitively inhibited anilinonaphthalene sulfonate binding to tubulin, and suppressed tubulin assembly. In cells, tubulin-TsAuNPs interactions were manifested as a disrupted microtubule network, defective reassembly of cold-disassembled microtubules, and induction of tubulin acetylation. Our data indicate that TsAuNPs inhibit cell viability by inducing differential cell cycle arrest possibly through disrupted dynamicity of cellular microtubules.

Bioassay-Guided Isolation and Evaluation of Antiproliferative Effects of (Z)-Ethylidene-4, 6-Dimethoxycoumaran-3-One from Pogostemon Quadrifolius (Benth.)

The aim of the study was to isolate and identify the major cytotoxic principle from plant leaves of Pogostemon quadrifolius (Benth.) and evaluate its antiproliferative potential against human cancer cells. Plant leaves were extracted sequentially with a soxhlet apparatus, using petroleum ether, chloroform and methanol solvents. Petroleum ether and chloroform extracts exhibited antiproliferative properties against Caco-2, HeLa, THP-1, MCF-7 and Jurkat E6-1cancer cell lines tested, but methanol extracts failed to exhibit such activity. The major antiproliferative principle from petroleum ether and chloroform extracts was isolated with the help of bioassay guided column chromatography. This cytotoxic compound was further analysed by UV, TLC, HPLC, LC-MS, GC-MS and NMR analyses and was identified to be novel: (Z)-ethylidene-4,6-dimethoxycoumaran-3-one (Compound 1). The half-maximal inhibitory concentrations for proliferation (IC50) exhibited by compound 1 were 19.4, 23.1, 22.1, 35.9 and 8.32 µM against Caco-2, HeLa, THP-1, MCF-7 and Jurkat E6-1 cancer cell lines, respectively. Further experiments revealed that compound 1 triggered the apoptosis mode of cell death in cancer cell lines. Thus, the present study allowed isolation and identification of a novel cytotoxic natural compound, (Z)-ethylidene-4,6-dimethoxycoumaran-3-one, from plant leaves of P. quadrifolius (Benth.). Our pre-clinical study also indicated that compound 1 is particularly active in the acute T cell leukemia cell line (Jurkat E6-1) with potential for application as a chemotherapeutic agent in the future.

Induction of acetylation and bundling of cellular microtubules by 9-(4-vinylphenyl) noscapine elicits S-phase arrest in MDA-MB-231 cells.

Noscapine is an alkaloid present in the latex of Papaver somniferum. It has been known for its anticancer efficacy and lack of severe toxicities to normal tissues. Structural alterations in noscapine core architecture have produced a number of potent analogues of noscapine. Here, we report an unusual activity of a novel noscapine analogue, 9-(4-vinylphenyl)noscapine (VinPhe-Nos) on cancer cells. As we reported earlier, VinPhe-Nos inhibited MDA-MB-231 cell proliferation with an IC50 of 6µM. The present study elucidated a possible antiproliferative mechanism of action of VinPhe-Nos. The noscapinoid significantly inhibited clonogenic propagation of MDA-MB-231 cells. However, unlike the majority of tubulin-binding agents, it did not induce mitotic arrest; instead, it prolonged S-phase. Although prolonged presence of the drug show some disruption of cellular microtubule architecture, it did not affect microtubule recovery after cold-induced depolymerization. VinPhe-Nos, nevertheless, induced acetylation and bundling of microtubules. Our data suggest that rational modification of parent compound can alter its mechanism of action on cell cycle and that VinPhe-Nos can be investigated further as a less-toxic, S-phase-preferred, cytostatic anticancer agent.

Dimethyl sulfoxide inactivates the anticancer effect of cisplatin against human myelogenous leukemia cell lines in in vitro assays.

OBJECTIVES: To investigate the effect of DMSO on cisplatin induced cytotoxicity (invitro) against K562 (Human mylogenous leukemia) cell line and to study the cisplatin-DMSO adduct formation using UV-spectrophotometer. MATERIALS AND METHODS: Effect of DMSO on the cytotoxicity of cisplatin was studied in K562 (Chronic mylogenous leukemia) cell line by MTT assay. Cisplatin-DMSO adduct formation was studied by continuously monitoring the increase in absorption peaks for 30 minutes using UV-spectrophotometer. RESULTS: 0.1-0.3% DMSO markedly reduced the cytotoxic activity of cisplatin in K562 cells. Cisplatin-DMSO adduct formation was detected using UV-spectrophotometer. Continuous increase in UV absorbance between 250nm-290nm was observed when cisplatin (0.5mg/ml) and DMSO (10%) were mixed. CONCLUSION: Present study revealed that DMSO inactivates the cytotoxicity of cisplatin. Cisplatin-DMSO mixture showed increased absorbance at 250-290nm. Therefore, using DMSO in invitro assays might result in misinterpretation of actual efficacy of drugs.

14-Deoxy-11,12-didehydroandrographolide inhibits proliferation and induces GSH-dependent cell death of human promonocytic leukemic cells.

14-Deoxy-11,12-didehydroandrographolide (AND2), an analogue of andrographolide, showed more potent cytotoxicity against human promonocytic leukemia (THP-1) cells than adherent cancer cell lines. In this study AND2 was isolated from the plant Andrographis paniculata and it was characterized. The antiproliferative effect of AND2 on both adherent (PC-3 and MDAMB) and non-adherent (THP-1 and Jurkat) cancer cell lines was evaluated by MTT assay. The effect of intracellular reduced glutathione (GSH) on AND2-induced cytotoxicity was studied by conducting cell viability assays on GSH-pretreated cells. The effect of AND2 on the redox status of THP-1 cells was determined by analyzing the endogenous reduced GSH content. Apoptosis induction was confirmed by DNA laddering assay and Western blot analysis using anti-caspase-3 protein antibody. AND2 showed antiproliferative action on both THP-1 and Jurkat cancer cell lines with low IC50 values. Cytotoxicity of AND2 was reversed by GSH pretreatment. AND2 treatment decreased the GSH content by 19.76 % (p < 0.001) in the THP-1 cancer cell line and reduced the cell clumping between the THP-1 cells. Expression of procaspase-3 varied in THP-1 cells during the time course of AND2 treatment. Procaspase-3 expression reached a maximum in treated cells at 32 h and was markedly reduced at 48 h but no procaspase-3 cleavage was observed. The obtained results suggest that AND2 is more effective against leukemia cells. AND2 induced a redox-mediated cell death in THP-1 cells. As AND2 temporarily increased the procaspase-3 expression during treatment, this study encourages the preclinical testing of AND2 against promonocytic leukemia cells in combination with small molecules that directly activate procaspase-3 to caspase-3.