Anti-tumorigenic Properties Research Articles

The Relative Expression of ERα Isoforms ERα66 and ERα36 Controls the Cellular Response to 24R,25-Dihydroxyvitamin D3 in Breast Cancer.

Vitamin D3 and its metabolites have antitumorigenic properties in vitro and in vivo; however, clinical trials and retrospective studies on the effectiveness of vitamin D3 oral supplementation against cancer have been inconclusive. One reason for this may be that clinical trials ignore the complex vitamin D metabolome and the many active vitamin D3 metabolites present in the body. Recent work by our lab showed that 24R,25(OH)2D3, a vitamin D3 metabolite that is active in chondrocyte proliferation and differentiation, has antitumorigenic properties in estrogen receptor alpha-66 (ERα66)-positive (ER+) breast cancer, but not in ERα66-negative (ER-) breast cancer. Here we show that 24R,25(OH)2D3 is protumorigenic in an in vivo mouse model (NOD.Cg-PrkdcscidIl2rgtm1Wjl /SzJ (NSG) mice) of ER- breast cancer, causing greater tumor growth than in mice treated with vehicle alone. In vitro results indicate that the effect of 24R,25(OH)2D3 is via a membrane-associated mechanism involving ERs and phospholipase D. 24R,25(OH)2D3 increased proliferation and reduced apoptosis in ERα66-negative HCC38 breast cancer cells, and stimulated expression of metastatic markers. Overexpressing ESRI, which encodes ERα66, ERα46, and ERα36, reduced the proapoptotic response of ERα66- cells to 24R,25(OH)2D3, possibly by upregulating ERα66. Silencing ESR1 in ERα66+ cells increased apoptosis. This suggests 24R,25(OH)2D3 is differentially tumorigenic in cancers with different ERα isoform profiles. Antiapoptotic actions of 24R,25(OH)2D3 require ERα36 and proapoptotic actions require ERα66. IMPLICATIONS: These results suggest that 24R,25(OH)2D3, which is a major circulating metabolite of vitamin D, is functionally active in breast cancer and that the regulatory properties of 24R,25(OH)2D3 are dependent upon the relative expression of ERα66 and ERα36.

Botanical characteristics, biological, chemical and medicinal properties of Rhus natalensis Bernh. ex. Krauss (Anacardiaceae)

Rhus natalensis Bernh. ex. Krauss is a prized medical shrub usually found in evergreen bushland and woodland, riverine associations, forest edges and frequently on well-drained gradients. The purpose of the current study was to assess existing literature on the botany, biological, chemical and medicinal properties of R. natalensis in an effort to determine the therapeutic potential of the species. Indigenous peoples from various ethnic groups in tropical Africa have used the plants for medicinal and other purposes. The majority of R. natalensis folkloric uses revolve around soreness, swelling and bacteriological infections. Furthermore, the leaves are often simmered and fed to cattle as a pain reliever. Several in vitro and in vivo analyses have shown that the plant possesses antibacterial, antifungal, antiviral, antimalarial, antioxidant, antifibrogenic, anti-inflammatory, antimutagenic, antithrombin, antitumorigenic, cytotoxic, hypoglycaemic and leukopenia properties. A total of seven phytochemical compounds have been isolated from R. natalensis which include tannins, flavonoids, biflavonoids, isoquinolines and terpenes, which shown various medicinal applications.

22β-hydroxytingenone induces apoptosis and suppresses invasiveness of melanoma cells by inhibiting MMP-9 activity and MAPK signaling

Ethnopharmacological relevance22β-hydroxytingenone (22-HTG) is a quinonemethide triterpene isolated from Salacia impressifolia (Miers) A. C. Smith (family Celastraceae), which has been used in traditional medicine to treat a variety of diseases, including dengue, renal infections, rheumatism and cancer. However, the anticancer effects of 22-HTG and the underlying molecular mechanisms in melanoma cells have not yet been elucidated. Aim of the studyThe present study investigated apoptosis induction and antimetastatic potencial of 22-HTG in SK-MEL-28 human melanoma cells. Materials and methodsFirst, the in vitro cytotoxic activity of 22-HTG in cultured cancer cells was evaluated. Then, cell viability was determined using the trypan blue assay in melanoma cells (SK-MEL-28), which was followed by cell cycle, annexin V-FITC/propidium iodide assays (Annexin/PI), as well as assays to evaluate mitochondrial membrane potential, production of reactive oxygen species (ROS) using flow cytometry. Fluorescence microscopy using acridine orange/ethidium bromide (AO/BE) staining was also performed. RT-qPCR was carried out to evaluate the expression of BRAF, NRAS, and KRAS genes. The anti-invasiveness potential of 22-HTG was evaluated in a three-dimensional (3D) model of reconstructed human skin. Results22-HTG reduced viability of SK-MEL-28 cells and caused morphological changes, as cell shrinkage, chromatin condensation, and nuclear fragmentation. Furthermore, 22-HTG caused apoptosis, which was demonstrated by increased staining with AO/BE and Annexin/PI. The apoptosis may have been caused by mitochondrial instability without the involvement of ROS production. The expression of BRAF, NRAS, and KRAS, which are important biomarkers in melanoma development, was reduced by the 22-HTG treatment. In the reconstructed skin model, 22-HTG was able to decrease the invasion capacity of melanoma cells in the dermis. ConclusionsOur data indicate that 22-HTG has anti-tumorigenic properties against melanoma cells through the induction of cell cycle arrest, apoptosis and inhibition of invasiveness potential, as observed in the 3D model. As such, the results provide new insights for future work on the utilization of 22-HTG in malignant melanoma treatment.

Path of Silibinin from diet to medicine: A dietary polyphenolic flavonoid having potential anti-cancer therapeutic significance.

In the last few decades, targeting cancer by the use of dietary phytochemicals has gained enormous attention. The plausible reason and believe or mind set behind this fact is attributed to either lesser or no side effects of natural compounds as compared to the modern chemotherapeutics, or due to their conventional use as dietary components by mankind for thousands of years. Silibinin is a naturally derived polyphenol (a flavonolignans), possess following biochemical features; molecular formula C25H22O10, Molar mass: 482.44 g/mol, Boiling point 793 °C, with strikingly high antioxidant and anti-tumorigenic properties. The anti-cancer properties of Silibinin are determined by a variety of cellular pathways which include induction of apoptosis, cell cycle arrest, inhibition of angiogenesis and metastasis. In addition, Silibinin controls modulation of the expression of aberrant miRNAs, inflammatory response, and synergism with existing anti-cancer drugs. Therefore, modulation of a vast array of cellular responses and homeostatic aspects makes Silibinin an attractive chemotherapeutic agent. However, like other polyphenols, the major hurdle to declare Silibinin a translational chemotherapeutic agent, is its lesser bioavailability. After summarizing the chemistry and metabolic aspects of Silibinin, this extensive review focuses on functional aspects governed by Silibinin in chemoprevention with an ultimate goal of summarizing the evidence supporting the chemopreventive potential of Silibinin and clinical trials that are currently ongoing, at a single platform.

Astragaloside IV Exerts Anti-tumor Effect on Murine Colorectal Cancer by Re-educating Tumor-Associated Macrophage.

Astragaloside IV (AS-IV) has shown anti-tumorigenic properties in certain cancers for its effect of boosting the body's immune system, but its role in colorectal cancer (CRC) remains unclear. In this study, we investigated the therapeutic effect of AS-IV in CRC and explored its underlying mechanism. CT26 colon cancer cells and mouse model by injection of CT26 cells subcutaneously were used as in vitro and in vivo model. M1 and M2 macrophage-associated markers, mRNA and protein expression levels were analyzed after AS-IV treatment. Inflammatory factors and cytokines in the tumors from mouse model were detected. Repolarization effect of AS-IV in vitro on bone-marrow-derived macrophages was also detected. In vitro, AS-IV inhibited the proliferation of CT26 cells and induced cell apoptosis dose-dependently, and significantly reduced M2 macrophages and increased M1 macrophages. In mouse model, it suppressed tumor growth and decreased the production of anti-inflammatory factors such as TGF-β, IL-10 and VEGF-A, while increased the production of pro-inflammatory factors like IFN-γ, IL-12 and TNF-α in tumor. Combination of AS-IV and checkpoint inhibitor aPD-1 exhibited synergistic antitumor effect by inhibiting tumor growth and increasing T cell infiltration. AS-IV could induce M2 macrophages polarization to the M1 phenotype. Its combination with immune checkpoint inhibitors could be expected to become a potential new strategy for the treatment of CRC.

Terpenes with Antitumor Activity: A Patent Review.

Cancer is a major public health concern, and is one of the leading causes of death globally. Surgical removal, chemotherapy or hormonal therapy, radiation therapy, or a combination of them are treatment for cancer, many of which are ineffective or have serious side effects. In view of this, there is a search for new, more effective alternatives for cancer prevention and treatment. One possible source of compounds are natural products; among them, terpenes, a large class of organic compounds, have shown promise due to their anti-inflammatory, anti-tumorigenic, and hypolipidemic properties, among others recorded in the literature. The study aims to use a patent review to evaluate the development and use of terpenes, or formulations containing terpenes, in new therapeutic options for the treatment of various types of cancer. This patent review was carried out using the specialized patent databases of WIPO and Espacenet. The selection of patents was based on the following inclusion criteria which included pre-clinical and/or clinical trials, and demonstrated anti-tumor effects. Eight patents were identified, six from China, and two from Japan. In this review, all patents confirmed having good antitumor activity for many types of cancer cells. In addition, the inventors indicate more studies pre-clinical and clinical trials giving greater clarity and accurate reflection of the activity of the products studied. Natural products are an important source of compounds for use in the fight against cancer that can act synergistically, and help in the treatment of cancer.

Cx43 and Associated Cell Signaling Pathways Regulate Tunneling Nanotubes in Breast Cancer Cells.

Simple SummaryThe gap junction protein connexin 43 (Cx43) facilitates direct intercellular communication and displays a complex dichotomous role in cancer. Here, we used breast cancer cell lines and CRISPR/Cas9 technology to dissect some of the poorly understood multimodal functions of Cx43 in this setting. Our data show that Cx43 directly modulates the formation of pro-tumorigenic tunneling nanotubes (TNTs), which are cellular protrusions that connect non-adjacent cells to facilitate cellular communication, including the direct cell–cell transfer of organelles. We identify several important cancer signaling pathways that affect TNT formation and show that this effect is strongly influenced by the presence or absence of Cx43. Finally, we demonstrate that Cx43 can affect TNT formation by modulating the cellular secretome. This work provides important insight into the pro-tumorigenic role of Cx43 and its interconnections with TNTs.Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that the loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by the inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, the drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type (WT) cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.

Synergistic effects of combined treatment with ultrasound-mediated cisplatin-loaded microbubbles and atorvastatin on head and neck cancer.

Previously, we used ultrasound (US)-mediated cisplatin (CDDP)-loaded microbubbles (CDDP-MBs) to increase intratumoral CDDP level while decreasing systemic cytotoxicity. Statins have shown antitumorigenic properties. Our study investigated the effects of atorvastatin with CDDP-MBs and US on head neck cancer. Cell viability analysis with CDDP-MBs and atorvastatin combined with US in FaDu cell line were tested. Cell proliferation and glutathione level were also evaluated. Both CDDP and atorvastatin reduced cell's viability. Coadministration of CDDP and atorvastatin resulted in synergistic inhibitory effect. After US sonication, cell viability with atorvastatin and CDDP was significantly reduced for CDDP combined with MBs (65.98% to 49.13%) and for CDDP-MBs (86.17% to 50.15%). CDDP-MBs combined with atorvastatin and US inhibited the proliferation of cells: 19.61% for CDDP-MBs + atorvastatin + US, 36.28% for CDDP + atorvastatin, and 71.73% for atorvastatin alone. Also, CDDP-MBs + atorvastatin + US induced apoptosis by decreasing cellular level of glutathione. Atorvastatin combined with MB-conjugated CDDP exerts synergistic inhibitory effect on head neck cancer.

Therapeutic effects of rosemary (Rosmarinus officinalis L.) and its active constituents on nervous system disorders.

Rosemary (Rosmarinus officinalis L.) is an evergreen bushy shrub which grows along the Mediterranean Sea, and sub-Himalayan areas. In folk medicine, it has been used as an antispasmodic, mild analgesic, to cure intercostal neuralgia, headaches, migraine, insomnia emotional upset, and depression. Different investigations have highlighted rosemary neuropharmacological properties as their main topics. Rosemary has significant antimicrobial, anti-inflammatory, anti-oxidant, anti-apoptotic, anti-tumorigenic, antinociceptive, and neuroprotective properties. Furthermore, it shows important clinical effects on mood, learning, memory, pain, anxiety, and sleep. The aim of the current work is to review the potential neuropharmacological effects of different rosemary extracts and its active constituents on nervous system disorders, their relevant mechanisms and its preclinical application to recall the therapeutic potential of this herb and more directions of future research projects. The data were gathered by searching the English articles in PubMed, Scopus, Google Scholar, and Web of Science. The keywords used as search terms were ‘Rosmarinus officinalis’, ‘rosemary’, ‘nervous system’, ‘depression’, ‘memory’, ‘Alzheimer’s disease’ ‘epilepsy’, ‘addiction’, ‘neuropathic pain’, and ‘disorders’. All kinds of related articles, abstracts and books were included. No time limitation was considered. Both in vitro and in vivo studies were subjected to this investigation. This review authenticates that rosemary has appeared as a worthy source for curing inflammation, analgesic, anti-anxiety, and memory boosting. It also arranges new perception for further investigations on isolated constituents, especially carnosic acid, rosmarinic acid, and essential oil to find exquisite therapeutics and support drug discovery with fewer side effects to help people suffering from nervous system disorders.

Complex effect of continuous curcumin exposure on human bone marrow-derived mesenchymal stem cell regenerative properties through matrix metalloproteinase regulation.

Curcumin has been reported to be beneficial for cancers, cardiovascular and neurodegenerative diseases, based on its anti-oxidative, anti-inflammation, anti-tumorigenic and neuroprotective properties. With its high-dose application, curcumin toxicity to systemic tissues is a reasonable concern. Here, we report the responses of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) to continuous curcumin exposure. hBM-MSCs were treated with 0.01-100μmol/L curcumin continuously in vitro for 7days. 25μmol/L curcumin or above significantly attenuated hBM-MSC maintenance, whereas 10μmol/L curcumin reduced hBM-MSC proliferation and hindered their migration with increasing cell apoptosis. Besides, 5μmol/L curcumin treatment inhibited hBM-MSC adipogenic differentiation, but enhanced osteogenic differentiation, which depended on matrix metalloproteinase (MMP)-13 expression and activity. Furthermore, curcumin treatment reduced MMP1 expression but up-regulated the immunomodulatory gene IDO1 expression. In summary, this study revealed the complex effects of continuous curcumin exposure on hBM-MSC maintenance and regenerative properties through MMP regulation. Given the complex effects of curcumin, its use for biomedical purposes should be carefully considered in treatment length and dosage.

Abstract 4023: Elevating the reactive oxygen species threshold in prostate cancer stem cells using VK3-OCH3 as a strategy to target prostate cancer

Abstract Prostate cancer (PCa) is the second most leading cause of cancer related deaths in American men. Although several cancer treatments are available, these have a number of drawbacks and can leave the patient with cardiotoxicity, loss of libido and infertility. Androgen deprivation therapy (ADT) is a standard promising therapy for metastatic PCa patients. However, patients within 18-24 months suffer from relapse and progress into castration resistant prostate cancer (CRPC). Experimental evidences suggest that, cancer stem cells (CSC) which constitute as a subpopulation of cancer cells, play a pivotal role in carcinogenesis, metastasis and chemoresistance to therapies. Therefore, targeting CSC can decrease the PCa recurrence rates and can help to offer better treatment options. Elevating the reactive oxygen species (ROS) threshold has been shown to be an effective strategy to eliminate CSC. Hence, in the present study, we hypothesized that VK3-OCH3, a menadione derivative, as a potential ROS enhancer in prostate cancer stem cell line (PCSC). After conducting a cell viability assay with different vitamin K (VK) analogs, our data revealed that VK3-OCH3 was more effective against PCSC compared to other VK analogs. Subsequently, colony formation assay and Trans-well migration assays revealed that VK3-OCH3 exhibits anti-tumorigenic and anti-metastatic properties against PCSC. Further studies showed that antioxidants such as NAC and GSH antagonized the effects of VK3-OCH3 on PCSC survival inhibition, suggesting the activation of oxidative stress by VK3-OCH3 in PCSC. In addition, our data revealed that VK3-OCH3 imparts oxidative stress by increasing the ROS levels which were determined using a ROS probe (DCFDA) by fluorescence analysis. VK3-OCH3 treatment also induced marked elevations in mitochondrial ROS generation in PCSC by FACS analysis which correlated with significant apoptotic cell death in these cells. Furthermore, we tested the effect of VK3-OCH3 on cell cycle progression using FACS and observed a significant G0 cell cycle arrest. By using an oncogene array, it was found that survivin, an apoptotic inhibitor, was downregulated by VK3-OCH3, which was further confirmed by Western blot analysis. Our subsequent confocal studies showed that, VK3-OCH3 downregulates the expression levels of EMT markers such as vimentin and TCF-8. Real time data also revealed that there is a modulation in gene expression of beta-catenin upon VK3-OCH3 treatment in PCSC. Of note, VK3-OCH3 treated PCSCs had a significant reduction in stem cell markers such as CD44 and ALDH1. In conclusion, our results suggest that VK3-OCH3 could be used as a potential ROS targeted anti-CSC strategy in treating PCa. Citation Format: Mona Vudutha, Somaiah Chinnapaka, Pranav Volety, Gnanasekar Munirathinam. Elevating the reactive oxygen species threshold in prostate cancer stem cells using VK3-OCH3 as a strategy to target prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4023.

Abstract 487: Safety and functional profiling of clinical grade mesenchymal stem cells derived from bone marrow, adipose tissue and Wharton's jelly for cancer therapy

Abstract Background: Mesenchymal stem cells (MSCs) have the potential as a drug delivery tool in cancer immunotherapy. We previously demonstrated that MSCs can be used to deliver BCG immunotherapy to the hypoxic core of tumors (#AACR-Abstract-3903). However, lack of clinical grade MSCs hamper the use of MSCs in clinical practice. The production of clinical grade human derived MSCs in accordance with current Good Manufacture Practice (cGMP) is an unmet need in the cancer immunotherapy. However, the selection criterion for characterizing the safety of MSC-based products is not well cited yet. In an effort to generate a potent therapeutic cell based product, we design a series of in-vitro safety assays for the production of high quality tissue specific human MSCs (hMSCs) as a final products before releasing from cGMP. Method: Here, we have isolated MSCs from Bone marrow (BM), Adipose tissue (AD) and Wharton Jelly (WJ) and expanded in serum free media. The in-vitro proliferation and phenotypic profiling of stem cell markers performed to check purity of the ex-vivo cultured hMSCs. To determine the clinical safety, we have done stemness marker analysis and cellular senescence capacity. To determine the functional aspects, tri-lineage differentiation and immunopotency of hMSCs were performed. Additionally, to assure the in vitro anti-tumorigenic properties of characterized hMSCs subjected to 2D colony forming unit (CFU) assay, 3D clonogenic assay as 3D organoid culture system and karyotyping. Results: Here, we proposed bioprocess design that allowed successful isolation of MSCs from BM, AD and WJ separately. Growth profiles of all isolated MSCs (n = 3) under serum free condition media with respect to serum containing media shows an exponential growth phase. The decline phase started after Day 7. Results also shows significant variation in population doubling time (PDT) of all isolated hMSCs at Passage 3 (n = 3) and Passage 5((n = 3) in comparison between serum free condition media & serum containing media. We found all hMSCs cultured in serum free media shows positive for CD105-APC (>80%), CD73-PE (>80%), CD90- PerCP (>80%), CD29- FITC (>80%) and HLAI (>80%), and negative HLAII- FITC(<2.20 %) and CD34/45- FITC (<2.20 %) expressions at Passage 3 & passage 5, indicating a stem cell phenotype (n = 3). Isolated hMSCs showed osteocyte, adipocytes and chondrocytes differentiation and high capacity to inhibit the proliferation of activated lymphocytes. Additionally, hMSCs positively expressed Nanog and Sox-2 stemness gene and cellular senescence from passage 7. In 2D CFU assay these cells showed colony forming capacity and in 3D organoid culture system in soft agar based matrix, we found that these cells are non-tumorigenic and didn't showed any karyotypic alteration. Conclusion: The collective result provides compelling evidence of the clinical safety of cGMP-compliant BM-MSCs, AD-MSCs and WJ-MSCs potentially suitable for future therapeutic application. Citation Format: Sorra Sandhya, Sonali Rawat, Suchi Gupta, Harshita Sharma, Bikul Das, Sujata Mohanty. Safety and functional profiling of clinical grade mesenchymal stem cells derived from bone marrow, adipose tissue and Wharton's jelly for cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 487.

Sonocomplexation as an effective tool to enhance the antitumorigenic effect of metformin: Preparation, in vitro characterization, molecular dynamic simulation & MiaPaCa-2 cell line hypoxia evaluation

This study aimed to prepare metformin-phospholipid sonocomplexes (MPS) to enhance the lipophilicity, hence the permeability of this highly water-soluble drug. Metformin (MET) is an old antidiabetic drug gaining interest for being recently investigated for its antitumorigenic properties. The polarity of MET makes its cellular uptake dependent on cell membrane transporters which poses a challenging limitation to combat cancer at supraphysiological concentrations unachievable in patients. A D-optimal design was adopted to statistically optimize the formulation variables, namely phospholipid: MET ratio, sonication time and phospholipid type. MPS showed a lipophilicity enhancement up to 19 folds based on the partition coefficient comparative study with pure MET. This vast improvement was explained using the packing parameter theory as we hypothesized the self-assembly of MPS in the lipid phase in the form of inverted micelles. This was confirmed by studying MPS on the molecular level using molecular docking and molecular dynamics simulation. The optimal sonocomplex showed 3.2 folds lower IC50, reduced oxygen consumption rate (OCR), hypoxia-inducible factor (HIF-1α) and reactive oxygen species (ROS) in MiaPaCa-2 cells compared to pure MET. These results revealed the potentiality of MPS to alleviate tumor hypoxia more effectively which could be useful for resistant cancers like pancreatic ductal adenocarcinoma (PDAC).

Anticancer Effects of Viscum album Fraxini Extract on Medulloblastoma Cells in vitro

Background: Mistletoe therapy is frequently administered as a supportive treatment in diverse pediatric cancer entities including brain tumors. Medulloblastoma is the most common brain tumor in childhood. Its high risk to metastasize and its long-term sequelae caused by aggressive chemo- or radiotherapies are still challenging. Material and Methods: Effects of a lectin-rich mistletoe extract, abnobaVISCUM Fraxini, were investigated in two medulloblastoma cell lines (Daoy and ONS-76). Responsiveness of tumor cells was assessed by cell viability assays and xCELLigence real-time analyses. Moreover, impacts on proliferation, cell cycle and apoptosis were investigated. Apoptosis was studied by staining of vital mitochondria and assessing the involvement of caspases. In addition, effects on migration and invasion were analyzed. Results: Both medulloblastoma cell lines were more susceptible to treatment with the mistletoe extract than a nontumorigenic fibroblast cell line. In mistletoe-sensitive Daoy cells, reduction of proliferation and induction of caspase-mediated apoptosis were observed upon administration of 0.05 and 0.5 mg/mL abnobaVISCUM Fraxini treatment, respectively. Furthermore, mistletoe extract inhibited migration and invasion properties in Daoy and significantly impaired invasive capabilities of ONS-76 cells. Conclusion: AbnobaVISCUM Fraxini has cell line dependent antitumoral effects in medulloblastoma models. These results call for further investigations, to reveal mechanistic insights into antitumorigenic properties of mistletoe extracts.

Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells

Human surfactant protein D (SP-D) belongs to the family of collectins that is composed of a characteristic amino-terminal collagenous region and a carboxy-terminal C-type lectin domain. Being present at the mucosal surfaces, SP-D acts as a potent innate immune molecule and offers protection against non-self and altered self, such as pathogens, allergens, and tumor. Here, we examined the effect of a recombinant fragment of human SP-D (rfhSP-D) on a range of breast cancer lines. Breast cancer has four molecular subtypes characterized by varied expressions of estrogen (ER), progesterone (PR), and epidermal growth factor (EGF) receptors (HER2). The cell viability of HER2-overexpressing (SKBR3) and triple-positive (BT474) breast cancer cell lines [but not of a triple-negative cell line (BT20)] was reduced following rfhSP-D treatment at 24 h. Upregulation of p21/p27 cell cycle inhibitors and p53 phosphorylation (Ser15) in rfhSP-D-treated BT474 and SKBR3 cell lines signified G2/M cell cycle arrest. Cleaved caspases 9 and 3 were detected in rfhSP-D-treated BT474 and SKBR3 cells, suggesting an involvement of the intrinsic apoptosis pathway. However, rfhSP-D-induced apoptosis was nullified in the presence of hyaluronic acid (HA) whose increased level in breast tumor microenvironment is associated with malignant tumor progression and invasion. rfhSP-D bound to solid-phase HA and promoted tumor cell proliferation. rfhSP-D-treated SKBR3 cells in the presence of HA showed decreased transcriptional levels of p53 when compared to cells treated with rfhSP-D only. Thus, HA appears to negate the anti-tumorigenic properties of rfhSP-D against HER2-overexpressing and triple-positive breast cancer cells.

A Novel Combination Treatment with Honokiol and Rapamycin Effectively Restricts c-Met-Induced Growth of Renal Cancer Cells, and also Inhibits the Expression of Tumor Cell PD-L1 Involved in Immune Escape.

The mTOR inhibitor Rapamycin has tumor inhibitory properties; and it is also used as an immunosuppressive agent after organ transplantation. However, prolonged Rapamycin treatment re-activates Akt and can promote cancer growth. Honokiol is a natural compound with both anti-tumorigenic and anti-inflammatory properties. Here, we assessed the anti-tumor effects of Rapamycin and Honokiol combination in renal cell carcinoma (RCC). Receptor tyrosine kinase c-Met-mediated signaling plays a major role in RCC growth. We observed that compared with Rapamycin alone, Rapamycin + Honokiol combination can effectively down-regulate c-Met-induced Akt phosphorylation in renal cancer cells; and it markedly inhibited Ras activation and cell proliferation and promoted G1 phase cell cycle arrest. The combination treatment significantly induced ROS generation and cancer cell apoptosis even when c-Met is activated. Importantly, Honokiol, but not Rapamycin, decreased c-Met-induced expression of the co-inhibitory molecule PD-L1, implied in the immune escape of renal cancer cells. In mouse renal cancer cells and Balb/c splenocytes co-culture assay, Rapamycin + Honokiol markedly potentiated immune-cell-mediated killing of cancer cells, possibly through the down-regulation of PD-L1. Together, Honokiol can effectively overcome the limitation of Rapamycin treatment alone; and the combination treatment can markedly restrict the growth of RCC, with particular importance to post-transplantation renal cancer.

Ciclopirox activates PERK-dependent endoplasmic reticulum stress to drive cell death in colorectal cancer

Ciclopirox (CPX) modulates multiple cellular pathways involved in the growth of a variety of tumor cell types. However, the effects of CPX on colorectal cancer (CRC) and the underlying mechanisms for its antitumor activity remain unclear. Herein, we report that CPX exhibited strong antitumorigenic properties in CRC by inducing cell cycle arrest, repressing cell migration, and invasion by affecting N-cadherin, Snail, E-cadherin, MMP-2, and MMP-9 expression, and disruption of cellular bioenergetics contributed to CPX-associated inhibition of cell growth, migration, and invasion. Interestingly, CPX-induced reactive oxygen species (ROS) production and impaired mitochondrial respiration, whereas the capacity of glycolysis was increased. CPX (20 mg/kg, intraperitoneally) substantially inhibited CRC xenograft growth in vivo. Mechanistic studies revealed that the antitumor activity of CPX relies on apoptosis induced by ROS-mediated endoplasmic reticulum (ER) stress in both 5-FU-sensitive and -resistant CRC cells. Our data reveal a novel mechanism for CPX through the disruption of cellular bioenergetics and activating protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent ER stress to drive cell death and overcome drug resistance in CRC, indicating that CPX could potentially be a novel chemotherapeutic for the treatment of CRC.

Serum Type XIX Collagen is Significantly Elevated in Non-Small Cell Lung Cancer: A Preliminary Study on Biomarker Potential.

Type XIX collagen is a poorly characterized collagen associated with the basement membrane. It is abnormally regulated during breast cancer progression and the NC1 (XIX) domain has anti-tumorigenic signaling properties. However, little is known about the biomarker potential of collagen XIX in cancer. In this study, we describe a competitive ELISA, named PRO-C19, targeting the C-terminus of collagen XIX using a monoclonal antibody. PRO-C19 was measured in serum of patients with a range of cancer types and was elevated in non-small cell lung cancer (NSCLC) (p < 0.0001), small cell lung cancer (p = 0.0081), breast (p = 0.0005) and ovarian cancer (p < 0.0001) compared to healthy controls. In a separate NSCLC cohort, PRO-C19 was elevated compared to controls when evaluating adenocarcinoma (AD) (p = 0.0003) and squamous cell carcinoma (SCC) (p < 0.0001) patients but was not elevated in chronic obstructive pulmonary disease patients. SCC also had higher PRO-C19 levels than AD (p = 0.0457). PRO-C19 could discriminate between NSCLC and healthy controls (AUROC:0.749 and 0.826 for AD and SCC, respectively) and maintained discriminatory performance in patients of tumor stages I+II (AUROC:0.733 and 0.818 for AD and SCC, respectively). Lastly, we confirmed the elevated type XIX collagen levels using gene expression data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) initiatives. In conclusion, type XIX collagen is released into circulation and is significantly elevated in the serum of cancer patients and PRO-C19 shows promise as a cancer biomarker.

Abstract C046: Dietary calcium and vitamin D and sun exposure with the risk of breast cancer among African American women

Abstract Background: Compared to European American women, African American (AA) women have a higher incidence of breast cancer (BrCa) under age 45, and are more likely to develop estrogen receptor-negative (ER-) tumors, a more aggressive BrCa subtype. Experimental studies drew attention to the potential antitumorigenic properties of calcium and vitamin D. Darker skin color reduces the cutaneous synthesis of vitamin D upon sun exposure. This, together with the tendency of AAs to consume less vitamin D and calcium from food and supplement sources, places AAs at risk for vitamin D and calcium deficiency. However, data are limited regarding the impact of calcium and vitamin D on risk of BrCa and specific subtypes among AA women. Objective: To evaluate calcium intake and vitamin D exposure (through food, supplements and sunlight) with the risk of BrCa and its subtypes among AA women. Methods: We evaluated these associations among 1033 BrCa cases and 391 controls of AA descent recruited since March 2012 in the Women's Circle of Health Study. Cases were identified by rapid case ascertainment in 10 counties in New Jersey. Controls were identified by random-digit-dialing and community-based recruitment, and were frequency matched by age group and county of residence. Dietary information over the year prior to diagnosis (for cases) or the reference date (for controls) was assessed via a validated food frequency questionnaire through in-person interviews. Supplemental intakes including multivitamin sources and daily hours spent outdoors in daylight were also collected. Hormone receptor status was obtained from pathology reports. Multivariable logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for relevant sociodemographic, reproductive, and lifestyle risk factors. Results: Vitamin D intake from food was not associated with BrCa risk. However, for supplemental vitamin D intake, we observed a significant decreased overall BrCa risk for users of ≤800 IU/day vs. nonusers (OR=0.73, 95% CI: 0.54, 0.98), which was limited to estrogen receptor-positive (ER+) BrCa (OR=0.69, 95% CI: 0.50, 0.94), but not for ER- or triple-negative BrCa. More daylight hours spent outdoors in a year predicted a lower risk of pre- and postmenopausal BrCa [comparing the highest quartile (Q4) vs. lowest (Q1): OR=0.37, 95% CI: 0.20-0.68, p-trend: 0.002; OR=0.46, 95% CI: 0.30-0.71, p-trend: 0.001, respectively]. The significant inverse associations were observed for both ER+ and ER- BrCa, but not for triple-negative BrCa. Calcium intake was not associated with BrCa risk. Conclusion: Our findings suggest that moderate intake of supplemental vitamin D may decrease the risk of ER+ BrCa. Sun exposure may decrease the risk of BrCa among AA women, which does not appear to differ by menopausal status and ER status. Citation Format: Bo Qin, Baichen Xu, Nan Ji, Karen Pawlish, Song Yao, Christine Ambrosone, Kitaw Demissie, Chi-Chen Hong, Elisa V. Bandera. Dietary calcium and vitamin D and sun exposure with the risk of breast cancer among African American women [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C046.

Antiproliferative effects of combinational therapy of Lycopodium clavatum and quercetin in colon cancer cells.

Background Colorectal cancer (CRC) is the third most prevalent form of cancer and fourth leading cause of morbidity worldwide. Surgical resection remains the only curative approach for CRC, but recurrence following surgery is the main problem and ultimate cause of death. Lycopodium clavatum and quercetin have been found to exert its anticancer properties. The aim of the present study is to investigate whether quercetin or L. clavatum extract and combination of both have any profound role in reducing major inflammatory cytokines in Colo-320 cells. Methods L. clavatum and Quercetin alone or in combination was administered to colon cancer cells and various toxicity markers, gene expression analyses of apototic genes and gelatin zymmography were performed. Results Quercetin (50 μm) in combination with L. clavatum extract (10 μL) distinctly reduced cell growth and highlighted their potential effects in extirpation of colon cancer cells. Treatment with increased dose of L. clavatum extract in combination with quercetin reduced the colony size and proliferation potential when compared to the sole treatment of plant extracts. In the antimicrobial assays, it was observed that Lycopodium alone exhibited antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Characterization of L. clavatum extract and quercetin was performed and confirmed the presence of flavonoids and alkaloids. Treatment with Lycopodium and quercetin combination induced significant down-regulation in activities of MMP2 and MMP9 tested by gelatin zymography. The combined treatment greatly affected the mRNA expression of p53, Bcl2, Bax, Caspase 3, Wnt 1, Cyclin D1, and Catalase genes in colon cancer cells. Conclusion The synergistic effect between Lycopodium and quercetin might bring forward the enhanced antitumorigenic properties of combinational therapy with natural products to successfully combat the cancer progression with minimal side effects and resistance to drugs.