Multivalency Effects: Example of Synthetic Ganglioside GM3 Analogues in the Study of Antitumor Agents.

Chemoenzymatically synthesized ganglioside GM3 analogues with inhibitory effects on tumor cell growth and migration

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), as an active ingredient in rhubarb roots and rhizomes, has been reported to possess various pharmacological properties including anti-tumor effects. Recent studies have confirmed that emodin inhibited cell proliferation and induced apoptosis of cancer cells. However, the inhibitory effect of emodin on the migration and invasion of melanoma cells and its underlying mechanism are still unclear. In the study, we observed the impercipient effects of emodin in B16F10 and A375 melanoma cells with strong metastatic abilities, focusing on the functions and mechanisms of migration and invasion of B16F10 and A375 melanoma cells. Cell counting kit-8 (CCK-8), colony formation test and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining tests confirmed that emodin possessed anti-proliferative and pro-apoptotic activities in B16F10 and A375 cells. The inhibitory effects on the migration and invasion of B16F10 and A375 cells were proved by wound healing assay and Transwell methods. Moreover, immunofluorescence assay approved the decrease in protein expression of matrix metalloproteinas (MMP)-2/-9 by emodin, and Western blot analyses revealed that emodin could increase the Bax/Bcl-2 ratio and inhibit the MMP-2/-9 protein expression and Wnt/β-catenin pathway in a dose-depended manner. BML-284, as an agonist of Wnt/β-catenin signaling pathway, reversed the effects of emodin on cell growth, migration and invasion in B16F10 cells. These findings may suggest that emodin treatment can be a promising therapeutic strategy for melanoma with highly metastatic abilities.

Objective To assess the effect of fenretinide (4-HPR) on proliferation, apoptosis and migration of B16F10 and A375 melanoma cells, and to evaluate the effect of liposomes and RGD peptide-modified liposomes on its uptake and therapeutic effects. Methods A film-hydration method was used to prepare 4-HPR liposomes (4-HPRL) , which were modified with RGD peptide to prepare RGD-4-HPRL, and the concentration, particle size, electric potential, drug loading capacity and encapsulation efficiency were measured for 4-HPRL and RGD-4-HPRL. In vitro cultured B16F10 and A375 cells were divided into several groups: 4-HPR group, 4-HPRL group and RGD-4-HPRL group treated with Dulbecco′s minimum essential medium (DMEM) containing 4-HPR bulk drug, 4-HPRL and RGD-4-HPRL respectively at the same concentration of 4-HPR, and control group treated with culture solution at the same volume. After different durations of treatment, cell counting kit-8 (CCK8) assay was performed to evaluate cellular proliferative activity, annexin V/propidium iodide staining to detect apoptosis, and scratch wound healing assay to evaluate the effect of drug treatment on cell migration ability. Then, 4-HPR was replaced by coumarin 6 (C6) to prepare C6 liposomes (C6L) and RGD-C6L, and flow cytometry was conducted to evaluate C6 uptake by B16F10 cells. Statistical analysis was carried out with SPSS22.0 software by one-way analysis of variance (ANOVA) for the comparison among several groups and t test for the comparison between two groups. Results The concentration of 4-HPR in the prepared 4-HPRL solution was over 1 300 mg/L. The encapsulation efficiency and drug loading capacity of 4-HPRL were (95.51 ± 1.22) % and (7.27 ± 0.11) % respectively, and those of RGD-4-HPRL were (95.82 ± 0.81) % and (7.14 ± 0.13) % respectively. The particle size distribution of 4-HPRL and RGD-4-HPRL was uniform, and their average particle size was below 100 nm. CCK8 assay showed that 4-HPR could markedly inhibit the proliferative activities of B16F10 and A375 cells. The cell proliferation inhibition rate of 4-HPRL was higher than that of 4-HPR at the same concentration of 4-HPR (P < 0.01) , and the inhibition rate of RGD-4-HPRL was higher than that of 4-HPRL (P < 0.01 or P < 0.05) and 4-HPR (P < 0.01) . As annexin V/propidium iodide apoptosis assay showed, when the concentration of 4-HPR was 10 mg/L, the total apoptosis rates of B16F10 cells in the control group, 4-HPR group, 4-HPRL group and RGD-HPRL group were (4.44 ± 0.35) %, (28.33 ± 0.66) %, (46.43 ± 0.77) % and (51.33 ± 0.37) % respectively. When the concentration of 4-HPR was 20 mg/L, the total apoptosis rates of A375 cells in the above 4 groups were (4.97 ± 0.62) %, (16.68 ± 3.81) %, (32.62 ± 1.24) % and (44.85 ± 4.92) % respectively. The apoptosis rates of B16F10 and A375 cells were significantly higher in the 4-HPRL group than in the 4-HPR group (both P < 0.01) , and higher in the RGD-4-HPRL group than in the 4-HPRL group (both P < 0.01) and 4-HPR group (both P < 0.01) . Scratch wound healing assay showed that 4-HPR could inhibit scratch healing and migration of B16F10 and A375 cells, and the inhibitory effects of 4-HPRL and RGD-4-HPRL were distinctly superior to those of 4-HPR bulk drug. C6 uptake assay revealed that the fluorescence intensity of C6 in B16F10 cells in the control group, C6 group, C6L group and RGD-C6L group were 2.15 ± 0.28, 8.56 ± 0.36, 20.48 ± 0.13 and 22.55 ± 0.07 respectively, and there were significant differences between the 4 groups (F = 67 194.186, P < 0.01) . Additionally, the fluorescence intensity of C6 was significantly higher in the C6L group and RGD-C6L group than in the C6 group (both P < 0.01) , and higher in the RGD-C6L group than in the C6L Group (P < 0.01) . Conclusions 4-HPR can inhibit the proliferation and migration of A375 and B16F10 cells, and induce their apoptosis. Liposomes and RGD-targeted liposomes can markedly enhance the effect of 4-HPR on melanoma cells. Key words: Melanoma; Cell line, tumor; Fenretinide; Liposomes; Integrin alphaVbeta3; Cell proliferation; Apoptosis; Cell migration assays

Alteronol inhibits the invasion and metastasis of B16F10 and B16F1 melanoma cells in vitro and in vivo

The Rho GDP dissociation inhibitor (RhoGDI) can bind to small GTPases and keep them in a biologically inactive state in cytoplasm, through which it affects actin polymerization and cell motility. However, mechanisms underlying how RhoGDI regulates Rho GTPase complex formation/membrane extraction/GTPase dissociation remain largely unexplored. Our previous studies reported that X-linked inhibitor of apoptosis protein (XIAP) interacted with RhoGDI via its RING domain and negatively modulated RhoGDI SUMOylation and HCT116 cancer cell migration. Here, we identified that RhoGDI SUMOylation specifically occurred at Lys-138, which was inhibited by XIAP domain. We further demonstrated that RhoGDI SUMOylation at Lys-138 was crucial for inhibiting actin polymerization and cytoskeleton formation as well as cancer cell motility. Moreover, SUMO-RhoGDI had a much higher binding affinity to small Rho GTPase compared with the un-SUMOylated form of RhoGDI. Taken together, our study demonstrated a novel modification of RhoGDI, SUMOylation at Lys-138, which played a key role in regulating Rho GTPase activation in cancer cells. The physiological regulation of RhoGDI SUMOylation by the RING domain of XIAP may account for modulation of cancer cell invasion and metastasis by XIAP.

Hydrogen sulfide (H2S) is widely recognized as the third endogenous gas signaling molecule and may play a key role in cancer biological processes. ADT-OH (5-(4-hydroxyphenyl)-3H-1,2-dithiocyclopentene-3-thione) is one of the most widely used organic donors for the slow release of H2S and considered to be a potential anticancer compound. In this study, we investigated the antimetastatic effects of ADT-OH in highly metastatic melanoma cells. A tail-vein-metastasis model was established by injecting B16F10 and A375 cells into the tail veins of mice, whereas a mouse footpad-injection model was established by injecting B16F10 cells into mouse footpads. We showed that administration of ADT-OH significantly inhibited the migration and invasion of melanoma cells in the three different animal models. We further showed that ADT-OH dose-dependently inhibited the migration and invasion of B16F10, B16F1 and A375 melanoma cells as evaluated by wound healing and Transwell assays in vitro. LC-MS/MS and bioinformatics analyses revealed that ADT-OH treatment inhibited the EMT process in B16F10 and A375 cells by reducing the expression of FAK and the downstream response protein Paxillin. Overexpression of FAK reversed the inhibitory effects of ADT-OH on melanoma cell migration. Moreover, after ADT-OH treatment, melanoma cells showed abnormal expression of the H2S-producing enzymes CSE/CBS and the AKT signaling pathways. In addition, ADT-OH significantly suppressed the proliferation of melanoma cells. Collectively, these results demonstrate that ADT-OH inhibits the EMT process in melanoma cells by suppressing the CSE/CBS and FAK signaling pathways, thereby exerting its antimetastatic activity. ADT-OH may be used as an antimetastatic agent in the future.

Aim: This study investigated the inhibition of proliferation and invasiveness of colorectal cancer (CRC) by niclosamide via targeting the secreted Frizzled-related protein 1 (SFRP1) and the Wnt/beta-catenin signaling pathway. Background: CRC is the third most common cancer worldwide. Aberrant activation of various signaling pathways in CRC leads to its poor response to chemotherapy. Hyperactivation of Wnt/beta-catenin pathway is known to drive cell proliferation, invasion, and migration of CRC. Epigenetic silencing of extracellular Wnt inhibitors, such as secreted Frizzled-related proteins (SFRPs), has been shown to stabilize beta-catenin and subsequently activate the Wnt signaling pathway. Niclosamide is a clinically approved drug indicated for the treatment of tapeworm infections. We have recently reported the repurposing of niclosamide to potentiate chemotherapeutic drugs for treating CRC by STAT3 inhibition. Niclosamide was also reported to be a Wnt/beta-catenin inhibitor but the precise mechanism is not clear. Methods: The antiproliferative effect was assessed by MTT assay and colony formation assay. The mRNA level, protein expression, and methylation status of SFRP1, and sets of SFRP1 co-expressed genes in CRC were analyzed by the TCGA data portal. The mRNA expression of SFRP1 and CpG island methylator phenotype (CIMP) marker genes were measured by qRT-PCR. DNA methylation was analyzed by bisulfite genomic sequencing. Inhibition of Wnt/beta-catenin and its downstream targets were examined by Western blot analysis. Cell migration and invasion were investigated by wound healing and transwell chamber migration assay. The intracellular localization of beta-catenin was determined by immunofluorescence assay. Results: Niclosamide inhibited HCT116 cell proliferation and colony formation in a concentration-dependent manner. Compared with normal colon tissues, CRC expressed significantly lower level of SFRP1 mRNA. SFRP1 methylation was inversely correlated with its mRNA expression. The DNA-demethylating agent 5-azacytidine was shown to restore expression of SFRP1 and CIMP panel genes in HCT116 cells after 5-day treatment. However, SFRP1 mRNA level was only upregulated by 1-day niclosamide treatment, but not 3-day niclosamide treatment. The upregulation of SFRP1 by niclosamide was found not relevant to DNA demethylation according to bisulfite genomic sequencing data. Immunofluorescence and Western blotting analysis showed that the increased SFRP1 expression was accompanied by reduced expression and nuclear accumulation of beta-catenin. Moreover, a low concentration of niclosamide that has minimal effect on cell proliferation, was shown to significantly suppress migration and invasion of HCT116 cells. Conclusion: Niclosamide is a promising candidate for drug repurposing. It was shown to upregulate SFRP1 and subsequently suppress Wnt/beta-catenin signaling, thereby impairing CRC cell migration and invasion. Upregulation of SFRP1 by niclosamide was not dependent on DNA demethylation. Citation Format: Mia Mingxia Wu, Christy Wing Sum Tong, Vivi Wei Yan, Kenneth Kin-wah To. Drug repurposing of niclosamide to regulate Wnt/beta-catenin signaling pathway through upregulating SFRP1 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB111.

Non-small cell lung cancer (NSCLC) accounts for the majority of lung cancer, with an unfavorable prognosis of 5-year survival rates. It is of great clinical significance to further search for more efficacious and novel targets for diagnosis and therapeutic strategies. This study aimed at clarifying the role of long non-coding RNA (lncRNA) NORAD in proliferation, invasion and migration and tumor growth of NSCLC. In this study, mRNA levels of lncRNA NORAD were examined by RT-PCR. CCK-8 assay was applied to test cell viability. Furthermore, wound healing assay and transwell assay were performed to detect the migration and invasion of A549 cells, respectively. Immunohistochemistry was applied to assess the levels of CXC chemokine receptor (CXCR) 4 and CXC chemokine ligand (CXCL) 12. Mice models of NSCLC in vivo were exploited to further examine the potential role of NORAD in tumor growth. Key proteins related to Ras homolog gene family member A (RhoA) GTPase/Rho-associated kinase (RhoA/ROCK) pathway were determined by Western blot. NORAD has elevated the levels in NSCLC tissues and cells. NORAD interference dramatically inhibited tumor growth and suppressed A549 cell proliferation, migration and invasion by downregulating CXCR4 and CXCL12 expression. RhoA/ROCK signaling pathway was activated in NSCLC. This study revealed that the downregulation of lncRNA NORAD could slow down the progression of NSCLC by regulating CXCR4 and RhoA/ROCK signaling pathway.

Background: Epithelial-mesenchymal transition (EMT) is considered to be a crucial event in the development of cancer metastasis. Metformin is an antidiabetic drug used in treatment of type 2 diabetes. Recently, increasing evidence has indicated that metformin can inhibit tumor growth, however, effect of metformin on EMT remains still well unknown. Methods: We investigated whether metformin inhibits EMT in the human pancreatic cancer cell lines (PANC-1, BXPC-3, and MIAPaCa-2). Pancreatic cancer cells were stimulated TGF-β1 (10ng/ml) with or without metformin(10mM). After 48h, we evaluated for EMT-related factors using western blot analyses, immunocytochemistry and RT-PCR. Wound healing assay was performed to determine cell migration. Results: The cells stimulated TGF-β1 acquired elongated and fusiform morphology, and metformin inhibited the TGF-β1-induced morphological changes in PANC-1 and BXPC-3 cells. Wound healing assay of PANC-1 and MIAPaCa-2 cells showed that significant wound healing was seen after 24h in cells treated with TGF-β1 compared with the control, and metformin inhibited wound closure. E-cadherin expression weakened after exposure to TGF-β1, but it was reversed by metformin in these cell lines. Metformin treatment reduced the expression of mesenchymal markers (Vimentin, N-cadherin and αSMA) induced by TGF-β1 in these cell lines. Exposure of cells to TGF-β1 resulted in the phosphorylation of mTOR, and exposure of cells to metformin resulted in the phosphorylation of AMPK and blocked the phosphorylation of mTOR in BXPC-3 cells. Furthermore, cell viability was no significantly change between exposing metformin or not. Discussion: Our results indicate that metformin inhibits TGF-β1 induced EMT in human pancreatic cancer cells. Moreover, our results suggest that metformin induces the phosphorylation of AMPK and inhibits TGF-β1 induced mTOR activation in BXPC-3 cells. Citation Format: Juichiro Yoshida, Takeshi Ishikawa, Yuki Endo, Shinya Matsumura, Takayuki Ohta, Katsura Mizushima, Yasuko Hirai, Tetsuya Okayama, Naoyuki Sakamoto, Ken Inoue, Kazuhiro Katada, Kazuhiro Kamada, Kazuhiko Uchiyama, Tomohisa Takagi, Yuji Naito, Yoshito Itoh. Metformin inhibits epithelial-mesenchymal transition in human pancreatic cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1038.

Design, synthesis and evaluation of 4,7-disubstituted 8-methoxyquinazoline derivatives as potential cytotoxic agents targeting β-catenin/TCF4 signaling pathway

Prosopis juliflora (Sw.) DC phytochemicals induce apoptosis and inhibit cell proliferation signaling pathways, EMT, migration, invasion, angiogenesis and stem cell markers in melanoma cell lines

Molecularly Engineered Aptamers Targeting Tumor Tissue and Cancer Cells for Efficient in Vivo Recognition and Enrichment

Background: The development of safe and effective novel agents remains a critical challenge for improving the prognosis of patients with unresectable colorectal cancer (CRC). Natural products containing polyphenols can be a good source of anti-cancer agents for their potential effect on multiple signaling pathways with anti-cancer effect. Due to its rich polyphenols content, Aronia berry extract (ABE) is gaining increased attention as an anti-cancer agent with its protective effect against cancer cells, including CRC, exerting potent antioxidant and antiproliferative effects. Thus, we hypothesized that ABE could be a novel complementary therapeutic option in patients with CRC by virtue of its ability to modulate multiple key cancer-related pathways. Methods: First, an MTT assay was performed to prove the antiproliferative effect of ABE in two CRC cells, SW480 and HCT116, and IC25 and IC50 of ABE in both CRC cells were identified. Next, the anti-cancer effects of ABE were examined by wound healing, colony formation, and invasion assays. Apoptosis studies were performed using the Muse Annexin V &amp; Dead Cell Assay Kit on the muse cell analyzer. RNA-sequencing was performed to clarify the critical regulatory genes and pathways modulated by ABE by comparing genome-wide profiling between CRC cells with and without ABE treatment. Results: Results of the MTT assay revealed that ABE decreased the viability of both CRC cells, and the IC25 and IC50 of ABE were 80.0 µg/mL and 130.0 µg/mL for SW480 and 80.0 µg/mL and 140.0 µg/mL for HCT116. ABE inhibited proliferation, migration, cellular survival, and invasion of CRC cells and induced apoptosis in those cells (SW480 control vs. IC25: p&amp;lt;0.05, control vs. IC50: p&amp;lt;0.01, HCT116 control vs. IC25: p&amp;lt;0.01, control vs. IC50: p&amp;lt;0.01). After ABE treatment, the genome-wide transcriptomic analysis identified 439 differentially expressed genes (p&amp;lt;0.05 and |log2FC|&amp;gt;0.5) in both CRC cells. These differentially expressed genes were used for pathway analysis and some critical cancer-related pathways, including the p53 signaling pathway (p=0.005, fold enrichment 3.8), NF-kappa B signaling pathway (p=0.030, fold enrichment 2.7), Hippo signaling pathway (p=0.037, fold enrichment 2.2), and MAPK signaling pathway (p=0.011, fold enrichment 2.0), were detected as significantly activated or suppressed pathways. Conclusions: We have investigated the anti-cancerous effect of ABE in CRC cells and identified multiple critical cancer-related pathways modulated by ABE, with the p53 signaling pathway as the key mediator for growth suppressive anti-cancer efficacy of this naturally occurring phytochemical. Our findings provide evidence for the use of ABE as a safe and effective complementary and integrative medicine approach against CRC. Citation Format: Yoh Asahi, Keisuke Okuno, Caiming Xu, Aknobu Taketomi, Ajay Goel. Novel evidence for the role of the p53 signaling pathway in mediating the anticancer effects of aronia berry extract in colorectal cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3822.

Sulfiredoxin (Srx) is multifunction enzyme with a primary antioxidant role of reducing the overoxidized inactive form of peroxiredoxins (Prxs). In contrast to the well-studied biochemical function, the biological significance of Srx in human diseases including cancer has not been well studied. Our previous studies show that it plays an important role in tumorigenesis and malignant progression of human skin, lung and colorectal cancers. Fascin homologue 1 (FSCN1) is an actin-binding protein that is critical for cancer cell invasion and metastasis. The purpose of this study is to understand the molecular mechanism of Srx on the invasion and metastasis of human colorectal cancer cells through the regulation of FSCN1. The effects of Srx on the migration and invasion of human colon cancer HCT116 cells were evaluated by wound healing and matrigel invasion assay, respectively. Actin stress fiber was visualized by F-actin fluorescence staining. Western blotting and reverse transcription &amp; real-time PCR were used to examine the protein and RNA levels of FSCN1 in HCT116 cells. The effect of Srx on FSCN1 promoter and 3’ UTR were determined using luciferase reporter construct. We demonstrated that (1) knockdown of Srx suppressed HCT116 colony formation and cell invasion in the matrigel invasion assay; (2) knockdown of Srx inhibited FSCN1 expression in HCT116 cells at both RNA and protein level; (3) the inhibition is the result of decreased FSCN1 mRNA stability; (5) Srx stabilizes FSCN1 mRNA 3’ UTR mediated reporter expression; (4) knockdown of Srx reduced actin stress-fiber formation; (5) Srx and Prx4 synchronically regulates FASN1 expression. Therefore, we concluded that Srx promotes the invasion and metastasis of human colon cancer cells through the upregulation of FSCN1. Citation Format: Hong Jiang, Qi Ying, B. Mark Evers, Qiou Wei. Sulfiredoxin promotes the invasion of human colorectal cancer cells through the activation of fascin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 177.

Nobiletin inhibits breast cancer cell migration and invasion by suppressing the IL-6-induced ERK-STAT and JNK-c-JUN pathways