Nitrosylcobalamin Selectively Targets Tumors via Cobalamin Uptake and Lysosomal Processing.

The ubiquitous plasma membrane transcobalamin II receptor (TC II-R) mediates uptake of cobalamin (Cbl; vitamin B12), an essential micronutrient. Tumors often require more Cbl than normal tissue, and increased Cbl uptake may result from increased TC II-R expression. To examine whether Cbl could therefore be used as a carrier molecule to target a chemotherapy drug, we tested an analogue of Cbl with nitric oxide as a ligand, nitrosylcobalamin (NO-Cbl). Because interferon beta (IFN-beta) has antitumor effects and increases expression of some membrane receptors, we examined whether it may enhance the effects of NO-Cbl. Antiproliferative effects of NO-Cbl were assessed in 24 normal and cancer cell lines. Xenograft tumors of human ovarian cancer NIH-OVCAR-3 cells were established in athymic nude mice, and tumor growth was monitored after treatment with NO-Cbl and IFN-beta, both individually and concomitantly. TC II-R expression and apoptosis was monitored in vitro and in vivo. RNA protection assays and mitochondrial membrane potential assays were used to distinguish the extrinsic and intrinsic apoptotic pathways, respectively. Cancer cell lines were more sensitive to NO-Cbl (with ID(50)s [the dose that inhibits growth by 50%] as low as 2 microM) than normal cell lines (with ID(50)s of 85-135 microM). Single-agent NO-Cbl and IFN-beta treatment of NIH-OVCAR-3 xenografts induced tumor regression, whereas combination treatment induced tumor eradication. IFN-beta treatment increased TC II-R expression in vitro and uptake of [(57)Co]cobalamin in vivo. Compared with NIH-OVCAR-3 cells treated with NO-Cbl, cells treated with NO-Cbl and IFN-beta were more apoptotic and expressed higher mRNA levels of various apoptosis-associated genes. No changes in mitochondrial membrane potential were observed in cells treated with NO-Cbl. NO-Cbl inhibited tumor growth in vivo by activating the extrinsic apoptotic pathway. The increased expression of TC II-R induced by IFN-beta resulted in enhanced antitumor effects with NO-Cbl both in vitro and in vivo.

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog.

We have previously demonstrated that nitrosylcobalamin (NO-Cbl), an analogue of vitamin B12 that delivers nitric oxide (NO), had potent antiproliferative activity against several human cancer cell lines. NO-Cbl induced apoptosis via a death receptor/caspase-8 pathway. In this study, we demonstrate that a functional Apo2L/TRAIL receptor was necessary for the induction of cell death by NO-Cbl. Furthermore, the Apo2L/TRAIL death receptor DR4 (TRAIL R1) was S nitrosylated following NO-Cbl treatment. Human melanoma (A375), renal carcinoma (ACHN), and ovarian carcinoma (NIH-OVCAR-3) cells were treated with NO-Cbl and subjected to the biotin switch assay; S-nitrosylated DR4 was detected in all three cell lines. NO-Cbl treatment did not cause S nitrosylation of DR5. The seven cysteine residues located in the cytoplasmic domain of DR4 were individually point mutated to alanines. NIH-OVCAR-3 cells expressing the DR4 C336A mutation lacked S nitrosylation following NO-Cbl treatment. Overexpression of wild-type DR4 sensitized cells to growth inhibition by NO-Cbl. Cells expressing the DR4 C336A mutant were more resistant to NO-Cbl and Apo2L/TRAIL than were the other six C-A mutations or wild-type cells. The C336A mutant also displayed blunted caspase-8 enzymatic activity following NO-Cbl treatment compared to the other mutants. Thus, DR4 residue C336 becomes S nitrosylated and promotes apoptosis following NO-Cbl treatment.

Chemoresistance is a major consequence of multicycle chemotherapy and can be attributed to constitutive activation of pro-survival signaling pathways. Nitric oxide is a ubiquitous signaling molecule which has been shown to inhibit several pathways involved with survival signaling in cancer cells. We have previously demonstrated the anti-tumor activity of a nitric oxide-donor, nitrosylcobalamin (NO-Cbl), mediated by increased expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. We also demonstrated that a functional Apo2L/TRAIL receptor is necessary for the induction of cell death by NO-Cbl and the Apo2L/TRAIL death receptor DR4 (TRAIL R1) is S-nitrosylated. The aim of the study was to examine the effects of nitric oxide (NO) on nuclear factor kappa B (NF-κB) and determine whether nitric oxide could sensitize drug-resistant melanomas to Apo2L/TRAIL via inhibition of NF-κB or inhibitor kappa B kinase (IKK). Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by the TUNEL assay. The activation status of NF-κB was established by assaying luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity. NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL, but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα. NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of nitric oxide to inhibit NF-κB and potentiate the effects of chemotherapeutic agents, such as Apo2L/TRAIL, represents a promising anti-cancer combination based on recent clinical investigations of anti-TRAIL antibodies for cancer treatment strategies.

Five compounds including quercetin (AC01), asparagine (AC02), sucrose (AC03), β-sitosterol-3-O-β-D-glucopyranoside (AC04) and β-sitosterol (AC05) were isolated from the methanol extract of Asparagus cochinchinensis (Lour.) Merr. tuber collected in Ba Ria–Vung Tau Province of Vietnam. Their structures were elucidated by NMR (1D and 2D-NMR). Quercetin (AC01) was subjected to the assay for antioxidant and anticancer activities. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay was employed for determining the antioxidant activity, while sulforhodamine B (SRB) method was applied for evaluating the anticancer activity against four selected human cancer cell lines. Quercetin had strong antioxidant activity with IC50 = 14.52 ± 2.12 µg/ml (as compared to standard vitamin C with IC50 = 10.49 ± 2.00 µg/ml). Meanwhile, quercetin (AC01) exhibited strong cytotoxicity against the HeLa, human cervical cancer cell line with IC50 = 5.78 ± 0.36 µg/ml, followed by lung cancer cell line (NCI–H460), lung cancer cell line with IC50 = 12.57 ± 1.19 µg/ml and liver cancer cell line (Hep-G2) liver cancer cell line with IC50 = 20.58 ± 0.85 µg/ml. The anticancer activity of quercetin against breast cancer cell line (MCF-7), breast cancer cell line was recorded with IC50 = 31.04 ± 3.14 µg/ml. Key words: Asparagus cochinchinensis, 1,1-diphenyl-2-picrylhydrazyl (DPPH), sulforhodamine B (SRB), human cervical cancer cell line (HeLa), lung cancer cell line (NCI-H460), breast cancer cell line (MCF-7), liver cancer cell line (Hep-G2).

Genes encoding growth-inhibitory proteins are postulated to be candidate tumor suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumors. Here we report the cloning and functional characterization of a novel human bone marrow stromal cell (BMSC)-derived growth inhibitor (BDGI) by large scale random sequencing of a human BMSC cDNA library. Human BDGI cDNA encodes a 477-amino acid residue protein that shares high homology with rat and mouse pregnancy-induced growth inhibitors. The C-terminal of BDGI is identical to a novel human pregnancy-induced growth inhibitor, OKL38. BDGI is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved family of BDGI-like proteins. BDGI overexpression inhibits the proliferation, decreases anchorage-dependent growth, and reduces migration of MCF-7 human breast cancer cells, whereas down-regulation of BDGI expression promotes the proliferation of MCF-7 and HeLa cervix epitheloid carcinoma cells. Interestingly, the inhibitory effect of BDGI on MCF-7 cells is more potent than that of OKL38. We demonstrate that BDGI induces cell cycle arrest in S phase and subsequent apoptosis of MCF-7 cells, which is likely to account for the antiproliferative effects of BDGI. This process may involve up-regulation of p27Kip1 and down-regulation of cyclin A, Bcl-2, and Bcl-xL. The inhibitory effect of BDGI on cell proliferation and the induction of apoptosis were also observed in A549 lung cancer cells but not HeLa cells. These results indicate that BDGI might be a growth inhibitor for human tumor cells, especially breast cancer cells, possibly contributing to the development of new therapeutic strategies for breast cancer.

Nitrosylcobalamin (NO-Cbl), a vitamin B12-based nitric oxide donor, has demonstrated selective antitumor activity. However, its translational development has been limited by formulation challenges, including restricted solution concentration and concerns regarding stability. To address these limitations, we developed a nanoemulsion reformulation of NO-Cbl (NanoE-NO-Cbl). NO-Cbl was reformulated into a nanoemulsion composed of soy lecithin, soybean oil, sucrose, and water using a high-shear Microfluidizer® processor. HPLC analysis and physicochemical characterization were used to assess NanoE-NO-Cbl for identity and stability across multiple storage conditions. Quantitative stability was evaluated by assay, chromatographic purity, pH, and droplet-size measurements. Antitumor activity was assessed in A375 melanoma cells using the sulforhodamine B (SRB) proliferation assay. Reformulation of NO-Cbl into a nanoemulsion increased the achievable formulation concentration relative to buffer-based preparations and enhanced potency approximately five-fold in A375 melanoma cells in vitro. NanoE-NO-Cbl demonstrated improved formulation stability under frozen and refrigerated storage conditions. Reformulation of NO-Cbl into a nanoemulsion improves formulation concentration, stability, and antitumor potency, providing a strong rationale for further preclinical development.

Intrinsic factor (IF) is a glycoprotein crucial for cobalamin (vitamin B12) absorption in the human body. This study aimed to evaluate the binding affinity of nitrosylcobalamin (NO-Cbl), a cobalamin analog, to recombinant human IF derived from plants, using hydroxocobalamin (OH-Cbl) as a comparative standard. Surface plasmon resonance (SPR) was employed to assess the kinetic parameters of NO-Cbl and OH-Cbl interactions with plant- derived IF across various concentrations. SPR analysis demonstrated that NO-Cbl and OH-Cbl exhibited high binding affinities to IF, with equilibrium dissociation constant (KD) values in the picomolar range. OH-Cbl showed a slightly stronger binding affinity (KD = 4.79 × 10-11 M) than NO-Cbl (KD = 8.58 × 10-11 M). Despite NO-Cbl and OH-Cbl both being bound to IF, differences in binding affinity and stability were observed, particularly at higher concentrations. Variations in IF binding between NO-Cbl and OH-Cbl may be attributed to the saturation of binding sites or recognition issues specific to plant-derived IF. This study underscores the potential of NO-Cbl as a targeted therapeutic agent capable of leveraging natural cobalamin uptake pathways. These results also highlight the suitability of using recombinant plant-derived IF as a model for predicting the biological activity of cobalamin analogs despite the nuanced differences from native human IF.

Cobalamin (Cbl; vitamin B12) is an essential micronutrient synthesized only by bacteria. Mammals have developed a sophisticated uptake system to capture the vitamin from the diet. Cbl transport is mediated by three transport proteins: transcobalamin, intrinsic factor, and haptocorrin (HC). All three proteins have a similar overall structure but a different selectivity for corrinoids. Here, we present the crystal structures of human HC in complex with cyanocobalamin and cobinamide at 2.35 and 3.0 Å resolution, respectively. The structures reveal that many of the interactions with the corrin ring are conserved among the human Cbl transporters. However, the non-conserved residues Asn-120, Arg-357, and Asn-373 form distinct interactions allowing for stabilization of corrinoids other than Cbl. A central binding motif forms interactions with the e- and f-side chains of the corrin ring and is conserved in corrinoid-binding proteins of other species. In addition, the α- and β-domains of HC form several unique interdomain contacts and have a higher shape complementarity than those of intrinsic factor and transcobalamin. The stabilization of ligands by all of these interactions is reflected in higher melting temperatures of the protein-ligand complexes. Our structural analysis offers fundamental insights into the unique binding behavior of HC and completes the picture of Cbl interaction with its three transport proteins.

In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin (like human IF, but unlike human HC and TC). The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared with human IF. The absorbance spectrum of the purified protein in complex with hydroxo-cobalamin resembled those of human HC and IF, but not TC. We searched available databases to further explore the phylogenies of the three cobalamin-binding proteins in higher vertebrates. Apparently, TC-like proteins are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis about a common ancestral gene for all cobalamin-binding proteins in higher vertebrates.

Background: In the last few decades, plants have been playing a vital role in treating cancer and infectious diseases. Natural products have been rediscovered as effective methods of drug production amid advances in combinatorial chemistry. Roots of Martynia annua are being used as a folklore remedy for the treatment of cancer and rheumatism successfully. Aims of the Study: In the present study, ethanolic, aqueous and hydro-ethanolic root extracts of Martynia annua were screened for in vitro cytotoxicity activity using different cell lines. Settings and Design: In the experiment, lung cancer cell lines (A549), leukemia cancer cell lines (K562), oral cancer cell lines (SCC-40), breast cancer cell lines (MCF-7) & cervix cancer cell lines (SiHa) were studied on the extracts. Materials and Methods: The method used was Sulforhodamine B (SRB) assay technique in which growth inhibition of 50% (GI50) was analyzed by comparing it with standard drug Adriamycin (ADR) (doxorubicin). Results: Aqueous & ethanolic extract of Martynia annua root had shown high anticancer activity with GI50 value 11.3µg/ml and 20.4µg/ml respectively on human leukemia cell line K-562 but for human breast cancer cell line MCF-7, human lung cancer cell line A-549, human squamous cell carcinoma SCC-40 and human cervical cancer cell line SiHa the extracts showed activity in more than 80µg/ml. Conclusion: The anticancer activity of aqueous extract of Martynia annua root was found superior than the ethanolic extract in Human Leukemia Cell Line K-562. The study indicates that the Martynia annua root extracts are most effective against the fast proliferative cells (Leukemic cells) and possibly a cell cycle arrest (needed to be proved as future perspective) is the mode of action of the extract. To study its effect on targeted cancers, specific in vivo scientific studies and clinical trials should be carried out by further researchers.

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins.

The sulforhodamine B (SRB) assay is a robust and cost-effective methods for assessing cellular proliferation and cytotoxicity in vitro. Originally designed to support drug screening efforts at the National Cancer Institute, this colorimetric assay quantifies protein content as a proxy for cell number and has proven reliable across diverse cancer cell lines. We present a fully standardized, step-by-step SRB assay protocol optimized for high-throughput application, educational accessibility, and translational relevance. Our streamlined protocol utilizes a fixed seeding density and simplified workflow, allowing for reproducible results with minimal technical expertise or specialized equipment. It has been successfully integrated into undergraduate teaching laboratories, while also supporting preclinical drug discovery efforts, including the identification of nitrosylcobalamin (NO-Cbl) as a novel anticancer agent. Key features of this protocol:•Provides an easy-to-follow format for generating reproducible cytotoxicity data in 96-well plates.•Can be used by novice researchers in teaching or early-stage discovery labs.•Suitable for scaling to support high-throughput compound screening campaigns.This accessible and validated SRB protocol has the potential to accelerate cancer research and empower future scientists at the bench.

Introduction: Omental metastasis is a defining feature of ovarian cancer and other cancers with peritoneal spread. We and others have previously shown that ovarian cancer (OvCa) cells readily home to the omentum in an adipokine-dependent manner and take up lipids from omental adipocytes to fuel metastasis. The complex interactions between omental adipocytes and OvCa cells, and the mechanisms by which OvCa cells adapt to this unique lipid-rich microenvironment, are still unclear and warrant further study. Here we used multiple omics platforms to study adipocyte-induced alterations in OvCa cells. Methods: We cocultured human primary adipocytes (HPA) with OvCa cell lines and then used mass spectrometry-based proteomic and global untargeted metabolomic analysis to study the altered cellular physiology of cancer cells upon encountering omental adipocytes. Stable cell lines were generated using lentivirus-mediated gene silencing and, subsequently, functional characterization was carried out using microarray and cell-based metabolic analysis. Since fatty acid binding protein 4 (FABP4) was identified in this analysis, we used a small-molecule inhibitor against FABP4 in an orthotopic xenograft mouse model to determine its role in promoting metastatic tumor burden. Results: Proteomic analysis revealed that HPA increases the expression of several proteins, especially those involved in lipid metabolism, such as cluster of differentiation 36 (CD36), FABP4, and alcohol dehydrogenase 1 (ADH1) in OvCa cancer cells. While we found that CD36 was essential for lipid uptake by the cancer cells, we determined that FABP4 was indispensable for their retention of intracellular lipid accumulation. Corollary metabolomic analysis showed that FABP4 knockdown dramatically reduced intracellular triacylglycerol levels induced by adipocyte coculture. We also found that FABP4 was responsible for the increased rate of β-oxidation observed in adipocyte cocultured cancer cells. Moreover, cancer cells exhibited increased oxidative stress when cultured with adipocytes or adipocyte conditioned media as evidenced by flow-cytometry analysis of reactive oxygen species. These observations were corroborated by detection of elevated oxidative stress markers such as oxidized lipids (15-HETE, 9-HODE, and 2-hydroxypalmitate), oxidized cholesterol (7-beta-hydroxy cholesterol), and oxidized glutathione. This increase in reactive oxygen species was found to be dependent on the levels of adipocyte-induced FABP4, suggesting that FABP4 plays a critical role in the alteration of cancer cells in contact with adipocytes. To determine the functional consequence of FABP4 inhibition in cancer cells, we carried out microarray and ingenuity pathway analysis (IPA) analysis after FABP4 knockdown, which revealed that FABP4 makes a significant contribution to proliferative and metastatic signatures in cancer cells. Knockdown of FABP4 also reduced colony-forming capacity in clonogenic assays and targeting FABP4 using a small-molecule inhibitor led to a significant reduction of metastatic burden in an orthotopic xenograft mouse model. Conclusions: Cancer cells in close proximity to adipocytes increase lipid-utilization genes. These genes enhance the ability of the cancer cells to use fatty acids, which are in abundance at the omentum, as an additional fuel source. FABP4, a lipid chaperon protein, was found to play a key role in regulating lipid utilization and redox-balance in cancer cells, helping them adapt to the omental microenvironment. Therefore, FABP4 may have potential as a therapeutic target against metastatic ovarian cancer. Citation Format: Abir Mukherjee, Fabian Coscia, Johannes Fahrmann, Chun-Yi Chiang, Justin Smith, Kristin Nieman, Andras Ladanyi, Iris Romero, Oliver Fiehn, Matthias Mann, Ernst Lengyel. Fatty acid binding protein 4 is indispensable for ovarian cancer metastasis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A15.

The main objective of the study was to evaluate the cytotoxicity of selected essential oils on human skin, gastric, and brain cancer cell lines using microculture tetrazolium test. Phytochemical analysis, as well as acute oral toxicity tests, was carried out in female albino mice with cardamom oil, lemon oil, and jasmine oil according to the Organization for Economic Co-operation and Development guidelines 425. Anticancer activities of the above test drugs were performed using human cancer cell lines. The studies were carried out at Skanda Life Sciences Pvt. Ltd., Bengaluru. Phytochemical analysis has shown the presence of carbohydrates and flavonoids in cardamom oil. While lemon oil has shown the presence of carbohydrates, flavonoids, steroids, terpenoids, and tannins, jasmine oil has shown the presence of carbohydrates, alkaloids, flavonoids, steroids, terpenoids, and glycosides. Toxicity studies showed that cardamom oil, lemon oil, and jasmine oil were all found to be safe up to 2000 mg/kg body weight. Results have shown that lemon oil exhibited the strongest cytotoxicity toward three human cancer cell lines, namely skin cancer (A431), gastric cancer (MKN-45), and brain cancer (U-87 MG) cell lines, with higher IC50 values of 62.82 μg/ml, 220.9 μg/ml, and 440.1 μg/ml compared to standard. Jasmine oil exhibited the strongest cytotoxicity toward skin cancer and brain cancer cell lines, whereas cardamom oil has shown stronger cytotoxicity only toward skin cancer cell line but did not show any level of inhibition of growth of brain and gastric cancer cells. Our study reveals that lemon oil, jasmine oil, and cardamom oil possess potent antitumor activity compared to standard. At different concentrations, lemon oil has shown statistically significant (***P < 0.0001) anticancer activity toward all the three human cancer cell lines. While jasmine oil has shown statistically significant (***P < 0.0001) anticancer activity toward skin and brain cancer cell line, cardamom oil has also shown statistically significant (***P < 0.0001) anticancer activity but only toward skin cancer cell line.