Anti-invasive Action Research Articles

Abstract 3809: Differential regulation of novel long non coding RNAs and their mechanism of action in p73 dependent manner

Abstract The p53 tumor suppressor family is classically activated after DNA damage and plays a central role in cell fate decisions. Although, the p53 family activates many of the same genes in response to DNA damage, p73 plays distinct biological functions in development and metastasis. It is likely that p73 activates a unique transcriptional network which is critical for its anti-metastatic and anti-invasive action. Long non-coding RNAs (lncRNAs) are a class of mRNA- like transcripts longer than 200 nucleotides. They lack protein-coding ability and are believed to be involved in various kinds of biological processes. Increasing evidence suggests that lncRNA are frequently aberrantly expressed in cancers. Therefore, the roles of dysregulated functional lncRNA in human malignant tumors have attracted considerable scientific interest. The objective of our study is to find out novel long non-coding RNAs that can act as transcriptional targets of p73 and to delineate their role in p73-mediated anti-metastatic response. For this purpose, we performed transcriptome sequencing in HCT116p73wt and HCT116p73KD cells and screened the data for modulation of expression of lncRNAs in differential manner. Quantitative Real Time PCR was further carried out to validate the data obtained after screening RNA seq Data. Promoter analysis was carried out for the identification of p73 binding sites in the selected upregulated or downregulated lncRNAs which was further confirmed by Luciferase reporter, ChIP and site directed mutagenesis assays. About six lncRNAs were observed to be significantly upregulated while four were down-regulated upon knockdown of p73. The promoters of selected lncRNAs were analysed in silico using TF Bind and JASPAR software for p73 binding sites and luciferase reporter assays suggested regulation of lncRNAs by p73. Chromatin immunoprecipitation showed promoter enrichment of the selected lncRNAs. Together, our study provides insights into the differential regulation of long non- coding RNAs in p73 dependent manner which further will provide the mechanism of their action at the genome level. Citation Format: Chanchal Bareja, Apoorva Uboveja, Daman Saluja. Differential regulation of novel long non coding RNAs and their mechanism of action in p73 dependent manner. [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 3809.

Abstract 1560: Elucidating the differential regulation of novel long non coding RNAs and their mechanism of action in p73 dependent manner

Abstract INTRODUCTION: The p53 tumor suppressor family is classically activated after DNA damage and plays a central role in cell fate decisions. Although, the p53 family activates many of the same genes in response to DNA damage, p73 plays distinct biological functions in development and metastasis. It is likely that p73 activates a unique transcriptional network which is critical for its anti-metastatic and anti-invasive action. Long non-coding RNAs (lncRNAs) are a class of mRNA-like transcripts longer than 200 nucleotides. They lack protein-coding ability and are believed to be involved in various kinds of biological processes. Increasing evidence suggests that lncRNA are frequently aberrantly expressed in cancers. Therefore, the roles of dysregulated functional lncRNA in human malignant tumors have attracted considerable scientific interest. The objective of our study is to find out novel long non-coding RNAs that can act as transcriptional targets of p73 and to delineate their role in p73-mediated anti-metastatic response. METHODS: For this purpose, we performed transcriptome sequencing in HCT116p73wt and HCT116p73KD cells and screened the data for modulation of expression of lncRNAs in differential manner. Quantitative Real Time PCR was further carried out to validate the data obtained after screening RNA seq Data. Promoter analysis was carried out for the identification of p73 binding sites in the selected upregulated or downregulated lncRNAs which was further confirmed by Luciferase reporter, ChIP and site directed mutagenesis assays. RESULTS: About six lncRNAs were observed to be significantly upregulated while four were down-regulated upon knockdown of p73. The promoters of selected lncRNAs were analysed in silico using TF Bind and JASPAR software for p73 binding sites and luciferase reporter assays suggested regulation of lncRNAs by p73. Chromatin immunoprecipitation showed promoter enrichment of the selected lncRNAs. Site directed mutagenesis further confirmed the exact binding sites of p73 onto the promoters of these novel long non coding RNAs. CONCLUSION: Together, our study provides insights into the differential regulation of long non-coding RNAs in p73 dependent manner which further will provide the mechanism of their action at the genome level. Citation Format: Chanchal Bareja, Apoorva Uboveja, Daman Saluja. Elucidating the differential regulation of novel long non coding RNAs and their mechanism of action in p73 dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1560.

Preimplantation factor modulates trophoblastic invasion throughout the decidualization of human endometrial stromal cells

BackgroundSuccessful human embryo implantation requires the differentiation of endometrial stromal cells (ESCs) into decidual cells during a process called decidualization. ESCs express specific markers of decidualization, including prolactin, insulin-like growth factor-binding protein-1 (IGFBP-1), and connexin-43. Decidual cells also control of trophoblast invasion by secreting various factors, such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases. Preimplantation factor (PIF) is a recently identified, embryo-derived peptide with activities at the fetal-maternal interface. It creates a favorable pro-inflammatory environment in human endometrium and directly controls placental development by increasing the human trophoblastic cells’ ability to invade the endometrium. We hypothesized that PIF’s effects on the endometrium counteract its pro-invasive effects.MethodsWe tested sPIF effect on the expression of three decidualization markers by RT-qPCR and/or immunochemiluminescence assay. We examined sPIF effect on human ESC migration by performing an in vitro wound healing assay. We analyzed sPIF effect on endometrial control of human trophoblast invasion by performing a zymography and an invasion assay.ResultsFirstly, we found that a synthetic analog of PIF (sPIF) significantly upregulates the mRNA expression of IGFBP-1 and connexin-43, and prolactin secretion in ESCs - suggesting a pro-differentiation effect. Secondly, we showed that the HTR-8/SVneo trophoblastic cell line’s invasive ability was low in the presence of conditioned media from ESCs cultured with sPIF. Thirdly, this PIF’s anti-invasive action was associated with a specifically decrease in MMP-9 activity.ConclusionTaken as a whole, our results suggest that PIF accentuates the decidualization process and the production of endometrial factors that limit trophoblast invasion. By controlling both trophoblast and endometrial cells, PIF therefore appears to be a pivotal player in the human embryo implantation process.

Cytotoxic activity of the seaweed compound fucosterol, alone and in combination with 5-fluorouracil, in colon cells using 2D and 3D culturing

ABSTRACTColorectal cancer (CRC) is one of the most frequently occurring carcinomas which require effective therapies. Fucosterol is a sterol present in marine brown seaweeds with several biological activities. However, the influence of fucosterol in CRC remains to be determined. Thus, the aim of this study was to examine the anticancer activity of fucosterol alone and in combination with 5-fluorouracil (5-Fu) on two human CRC cell lines (HCT116 and HT29) and compared with cytotoxicity in one normal colon fibroblast cell line (CCD-18co) in monolayer (2D). The effect of fucosterol alone or in combination with 5-Fu was further assessed using HT29 multicellular spheroids (3D). Data demonstrated that fucosterol alone or combined with 5-Fu decreased cell viability in HT29 cells in 2D cultures without inducing cytotoxic in normal colon cells. The combination, fucosterol, and 5-Fu, also inhibited cell proliferation, clonogenic potential and cell migration without producing cell death in 2D. In multicellular spheroids, the combination fucosterol plus 5-Fu at the same concentrations used in 2D was not effective demonstrating that under the tested conditions the 3D model was more resistant than the 2D model. Taken together, these findings suggest that fucosterol might be a promising alternative to enhance the cytotoxic and anti-invasive actions of 5-Fu in colon cancer cells without consequent major adverse effects in normal cells. Our results also reinforce the need to include more complex 3D culture models in the initial stages of drug screening.

Abstract 2872: Mechanism of anti-proliferative and anti-invasive actions of ω3-polyunsaturated fatty acids in human hypopharyngeal carcinoma cells

Abstract Hypopharyngeal carcinoma is one of head and neck cancers. According to the latest statistics from the United States National Cancer Institute, it is estimated that about 17,000 Americans will be diagnosed with pharyngeal cancer in 2017. Although ω3-polyunsaturated fatty acids (ω3-PUFAs) have anti-tumorigenic properties in the several cancers, the anti-cancer effect of ω3-PUFAs on hypopharyngeal carcinoma has been not known yet. In this study, we report inhibitory mechanisms of ω3-PUFAs on cell growth and invasion in hypopharyngeal carcinoma. DHA and EPA inhibited the cell growth of FaDu and SNU1041 in a dose- and time-dependent manner; in contrast, arachidonic acid, a ω6-PUFA, had no significant effect. In flow cytometry analysis, subG1 population increased in DHA-treated FaDu and SNU1041. Moreover, apoptotic cell death was confirmed by TUNEL assay, and the induction of cleaved PARP and caspase-3. Furthermore, treatment of hypopharyngeal carcinoma cells with DHA resulted in a significant increase in autophagic activity, as revealed by increased LC3-II levels, GFP-LC3 puncta, and autophagic flux activation. EGF-induced phosphorylation of EGFR, which is frequently overexpressed in hypopharyngeal carcinoma, was also suppressed after DHA pretreatment, and levels of p-Akt (p- Aktthr308 and AktSer473), which is a downstream signal, was also inhibited in the FaDu and SNU1041 cells. The invasiveness of cells was significantly inhibited by DHA treatment in vitro transwell assay as well. The MMP-2 and MMP-9 promoter activities were inhibited after DHA treatment. Cox-2 and VEGF promoter activities were also suppressed by DHA. Furthermore, DHA decreased the levels of reporter activity of NF-κB, which is transcription factor that regulates MMPs, Cox-2 and VEGF expression. Fat1-stably expressing FaDu cells (fFaDu-sc) was established (fFaDu-sc express a Caenorhabditis elegans ω3-desaturase converting ω6- to ω3-PUFAs endogenously), and the proliferation of fFaDu-sc was more attenuated than that of cells expressing control vectors (fFaDu-cc). Moreover, fFaDu-sc showed reduction of cell invasion compared with fFaDu-cc. in transwell chamber. The MMPs promoter activities were also suppressed in fFaDu-sc. Taken together, these findings provide evidence that ω3-PUFAs may inhibit invasion as well as cell growth through suppression of p-EGFR and MMPs expression in hypopharyngeal carcinoma cells, indicating that the utilization of ω3-PUFAs may represent a potential effective therapy for the chemoprevention and treatment of human hypopharyngeal carcinoma. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)]. Citation Format: Seung-Hyeon Han, Soyeon Shin, Young-Joo Jeon, Jun-Young Heo, Gi-Ryang Kweon, Seung-Kiel Park, Jong-Il Park, Kyu Lim. Mechanism of anti-proliferative and anti-invasive actions of ω3-polyunsaturated fatty acids in human hypopharyngeal carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2872.

All-trans-retinoic acid activates the pro-invasive Src-YAP-Interleukin 6 axis in triple-negative MDA-MB-231 breast cancer cells while cerivastatin reverses this action

All-trans-retinoic acid (RA), the active metabolite of vitamin A, can reduce the malignant phenotype in some types of cancer and paradoxically also can promote cancer growth and invasion in others. For instance, it has been reported that RA induces tumor suppression in tumor xenografts of MDA-MB-468 breast cancer cells while increasing tumor growth and metastases in xenografts of MDA-MB-231 breast cancer cells. The signaling pathways involved in the pro-invasive action of retinoic acid remain mostly unknown. We show here that RA activates the pro-invasive axis Src-YAP-Interleukin 6 (Src-YAP-IL6) in triple negative MDA-MB-231 breast cancer cells, yielding to increased invasion of these cells. On the contrary, RA inhibits the Src-YAP-IL6 axis of triple-negative MDA-MB-468 cells, which results in decreased invasion phenotype. In both types of cells, inhibition of the Src-YAP-IL6 axis by the Src inhibitor PP2 drastically reduces migration and invasion. Src inhibition also downregulates the expression of a pro-invasive isoform of VEGFR1 in MDA-MB-231 breast cancer cells. Furthermore, interference of YAP nuclear translocation using the statin cerivastatin reverses the upregulation of Interleukin 6 (IL-6) and the pro-invasive effect of RA on MDA-MB-231 breast cancer cells and also decreases invasion and viability of MDA-MB-468 breast cancer cells. These results altogether suggest that RA induces pro-invasive or anti-invasive actions in two triple-negative breast cancer cell lines due to its ability to activate or inhibit the Src-YAP-IL6 axis in different cancer cells. The pro-invasive effect of RA can be reversed by the statin cerivastatin.

Adiponectin limits differentiation and trophoblast invasion in human endometrial cells.

Successful human embryo implantation requires a proper differentiation of endometrial stromal cells (ESCs) into decidual cells, during a process called decidualization. ESCs express specific molecules, such as prolactin, insulin-like growth factor-binding protein-1 (IGFBP-1) and connexin-43. Decidual cells are also involved in the control of trophoblast invasion, by secreting various factors, such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Adiponectin is an adipokine with insulin-sensitizing, anti-inflammatory and anti-proliferative effects. At the embryo-maternal interface, adiponectin promotes differentiation and invasion of human trophoblastic cells. We hypothesize that the effects of adiponectin on endometrium could counteract its pro-invasive effects previously described in the human trophoblast. In this context, we have firstly demonstrated that adiponectin downregulates IGFBP-1 and connexin-43 mRNA expressions, as well as prolactin secretion in ESCs, suggesting an anti-differentiative effect of adiponectin. Secondly, we found that invasive capacities of trophoblastic cell line HTR-8/SVneo are reduced in the presence of conditioned media from ESC cultured in the presence of adiponectin. Adiponectin's anti-invasive action is associated with a decreased activity of MMP-2 and MMP-9, and an increased TIMP-3 mRNA expression in ESCs. Finally, adiponectin receptors (ADIPOR1 and ADIPOR2) knockdown abolishes the anti-differentiative and anti-invasive effects of adiponectin in human ESCs. Altogether, our results suggest that adiponectin reduces the decidualization process and inversely induces the production of endometrial factors that limit trophoblast invasion. Thus, through a dual control in trophoblast and endometrial cells, adiponectin appears as a pivotal actor of the embryo implantation process.

Abstract 2110: Anti-invasive and antimetastatic actions of omega-3 polyunsaturated fatty acids in glioblastoma cells

Abstract Glioblastoma (GBM) is the most aggressive and deadliest brain malignancy in adults. Despite of surgical techniques, radiotherapy and chemotherapy, GBM has remained an invariably lethal tumor with a median survival less than 15 months. Although the anticancer mechanisms of omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been reported in several cancers, it is still unclear in brain cancer. Here, our data show the anti-invasive and anti-metastatic effects of ω3-PUFAs in GBM. Invasiveness using transwell chamber was inhibited by docosahexaenoic acid (DHA) treatment in D54MG and GL261 cells. In zymography, MMP-2 activity was suppressed by DHA in a dose dependent manner. MMP-2 promoter activity was also decreased. DHA inhibited both p-STAT3 and β-catenin levels that contribute to invasion by stimulating pro-invasion factors such as MMP-2. Additionally, fat-1 (ω3-desaturease) stable D54MG cell was established and the effect of the high level of ω3-PUFAs was investigated endogenously. The invasiveness were significantly inhibited in the fat-1 stable cells compared to control cell. Moreover, the metastasis in vivo was significantly reduced when GL261 mouse glioma cells were injected through tail vein into the fat-1 transgenic mice. In immunohistochemistry, intensity of p-STAT-3 and β-catenin were significantly weak in metastatic lung tumor from Fat-1 mice compared to wild type mice. These results indicate that anti-invasive and anti-metastatic actions of ω3-PUFA may be correlated with inhibition of MMP-2 through decrease of STAT3 and β-catenin in GBM cells. Thus, these findings provide important preclinical evidence and molecular insight for utilization of ω-3 PUFAs for the chemoprevention and treatment of human GBM. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932, NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)]. Citation Format: Soyeon Kim, Soyeon Shin, Soyeon Jeong, Seung-Hyeon Han, Yoon-Seon Yoo, Prashanta Silwal, Young-Joo Jeon, Jun-Young Heo, Gi-Ryang Kweon, Seung-Kiel Park, Jong-Il Park, Kyu Lim. Anti-invasive and antimetastatic actions of omega-3 polyunsaturated fatty acids in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2110. doi:10.1158/1538-7445.AM2017-2110

Apoptotic and Anti-invasive Effects of Hybrid Liposomes without Drugs against Human Breast Cancer Cells In Vitro

Hybrid Liposomes (HL) composed of 90 mol% L-α-dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylene (25) dodecyl ether (C12(EO)25) was examined to study the stimulation of apoptosis and antiinvasive actions against in vitro cultured human breast cancer (MCF-7) cells. The results obtained from the study reveals that HL suppressed the growth of MCF-7 cells without any drugs. Apoptosis for MCF-7 cells after subjecting them to HL treatment was noticed. To study the non-invasive actions of HL on the drifting of MCF-7 cells, we exercised Wound scratch assay. Non-invasive effects of HL on the migration of MCF-7 cells were obtained on the basis of wound scratch assay. HL suppressed the pseudopodium formation of MCF-7 cells. The study to determine the non-invasive effects of HL against MCF-7 cells were done by matrigel invasion assay which gave positive results due to inhibition of MT1MMP/MMP14 by HL.

Abstract 1301: Anti-invasive and antimetastatic effects of ω3-polyunsaturated fatty acids through inhibition of NF-kB/matrix metalloproteinases in ovarian cancer cells

Abstract Ovarian cancer is leading cause of gene cological cancer death in the United States. It is reported that about 14,000 patients will die to ovarian cancer in United States, 2015. ω3- and ω6-PUFAs are considered as essential fatty acids because they cannot be synthesized in mammals. The ω3-desaturase (fat-1) converts ω6-PUFAs to ω3-PUFAs. Cancer cells and transgenic mice stably expressing fat-1 geneare useful models to study the anti-cancer effects of ω3-PUFAs. Here, we show anti-invasive and anti-metastatic action of ω3-PUFA in ovarian cancer.DHA significantly inhibited cell invasion and wound healing activity of human ovarian PA-1 cells. DHA also abolishedthe induction of matrix-metalloproteinases (MMPs) and cyclooxygenase (COX2) as well as the transactivity of NF-κB in PA-1 cells. When ID8 cells were subcutaneously implanted into fat-1 transgenic mice, the tumor size and volume were smallin comparison to wild-type mice. The growth inhibition of tumor was also confirmed with the increase in the level of TUNEL-positive cells. Additionally, when ID8 cells were injected via tail vein of fat1 mice, the lung metastasis was remarkably inhibited in fat-1 transgenic mice. Furthermore, the proliferation of fat-1-stably expressing PA-1 (f-PA-1) cells was more attenuated than that of cells expressing control vectors. The invasion and NF-κB/MMPs promoter activities were also decreased in f-PA-1 cells. These results suggest that ω3-PUFAs inhibit invasion and metastasis of ovarian cancer cells through inhibition of NF-κB/MMPs. Therefore, ω3-PUFAs may contribute an effective and safe therapeutic approach for the chemoprevention and treatment of human ovarian cancer. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932)] Citation Format: Soyeon Jeong, Kaipeng Jing, Soyeon Shin, Soyeon Kim, Young-Joo Jeon, Jun-Young Heo, Gi-Ryang Kweon, Seung-Kiel Park, Jong-Il Park, Kyu Lim. Anti-invasive and antimetastatic effects of ω3-polyunsaturated fatty acids through inhibition of NF-kB/matrix metalloproteinases in ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1301.

Fatty acid amide hydrolase inhibitors confer anti-invasive and antimetastatic effects on lung cancer cells.

Inhibition of endocannabinoid degradation has been suggested as tool for activation of endogenous tumor defense. One of these strategies lies in blockade of fatty acid amide hydrolase (FAAH) which catalyzes the degradation of endocannabinoids (anandamide [AEA], 2-arachidonoylglycerol [2-AG]) and endocannabinoid-like substances (N-oleoylethanolamine [OEA], N-palmitoylethanolamine [PEA]). This study addressed the impact of two FAAH inhibitors (arachidonoyl serotonin [AA-5HT], URB597) on A549 lung cancer cell metastasis and invasion. LC-MS analyses revealed increased levels of FAAH substrates (AEA, 2-AG, OEA, PEA) in cells incubated with either FAAH inhibitor. In athymic nude mice FAAH inhibitors were shown to elicit a dose-dependent antimetastatic action yielding a 67% and 62% inhibition of metastatic lung nodules following repeated administration of 15 mg/kg AA-5HT and 5 mg/kg URB597, respectively. In vitro, a concentration-dependent anti-invasive action of either FAAH inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Using siRNA approaches, a causal link between the TIMP-1-upregulating and anti-invasive action of FAAH inhibitors was confirmed. Moreover, knockdown of FAAH by siRNA was shown to confer decreased cancer cell invasiveness and increased TIMP-1 expression. Inhibitor experiments point toward a role of CB2 and transient receptor potential vanilloid 1 in conferring anti-invasive effects of FAAH inhibitors and FAAH siRNA. Finally, antimetastatic and anti-invasive effects were confirmed for all FAAH substrates with AEA and OEA causing a TIMP-1-dependent anti-invasive action. Collectively, the present study provides first-time proof for an antimetastatic action of FAAH inhibitors. As mechanism of its anti-invasive properties an upregulation of TIMP-1 was identified.

Potion of Poison? Cannabis oil.

Cannabis oil is a resinous substance extracted from Cannabis sativa or Cannabis indica. Obtained by separating the resins from cannabis flowers using isopropyl alcohol, cannabis oil is increasingly being sought after for its purported anticancer activity. PREPARATIONS: Cannabis oil can be taken orally, inhaled by vaporisation, applied topically or formulated into suppositories. High quality oil has a high percentage of active constituents, and is dependent on the cannabis species, quality of raw plant material, and the extraction procedure. COMMON NAMES: Marijuana oil, hash oil, pot oil, CBD oil, Rick Simpson Oil (RSO). LATIN NAME: Cannabis sativa and C. indica belong to the Cannabaceae family. ACTIVE CONSTITUENTS: The cannabis plant contains over 500 compounds including flavonoids, terpenoids and cannabinoids. Cannabinoids are the active compounds, of which delta-9-tetrahydrocannabinol (THC), the main psychoactive compound, and cannabidiol (CBD), which counteracts the psychoactive effect of THC, are produced in the highest concentration. MEDICAL CLAIMS: There is a growing interest, as well as numerous unsubstantiated claims, that cannabis and cannabinoids (especially in the high doses found in oil extraction) may kill cancer cells. It is claimed that this is through action on cannabinoid (CB1 and CB2) receptors and results in direct induction of apoptosis, direct inhibition of tumour growth, and inhibition of tumour angiogenesis and metastasis. EVIDENCE: An early clinical trial whose primary end point was to determine the safety of highly purified THC administered intracranially in patients with glioblastoma multiforme, showed possible antiproliferative actions on tumour cells. However, owing to obvious legal reasons, virtually all research has been in vitro or in animal models. Thus far, the best laboratory results have come from using a combination of highly purified THC and CBD, which have shown to exert anti-proliferative, pro-apoptotic, anti-migratory and anti-invasive actions in a variety of cancers. Conversely, evidence suggests that cannabinoids may encourage cancer cell growth, or have different effects depending on the receptors present on the cancer cell. Resistance to cannabinoids is also likely. More human trials are needed to understand exact mechanisms and signalling by which cannabinoids function. ADVERSE EFFECTS: Cannabinoids are considered to have a favourable safety profile. The lethal dose in animal studies is estimated to be several grams per kilogram. Most reported adverse effects in trials were fatigue and dizziness. Tachycardia, hypotension, decreased gastrointestinal motility, and muscle relaxation may occur as well as CNS effects (stimulation and depressing); however tolerance to unwanted effects develops quickly. Synthetic cannabinoids are advised not to be used in patients with a history of cardiovascular or psychotic disorder, children under 18 years, women who are pregnant and/or breastfeeding, and women and men who are planning a family. DRUG INTERACTIONS: Drug-drug and drug-disease interactions are largely unknown due to few human trials available. There may be an additive effect when used concomitantly with medication that affects heart rate (eg anticholinergics and tricyclic antidepressants) and alertness (benzodiazepines, opiates, alcohol). Studies show cannabinoids may induce CYP1A2, CYP2C and CYP3A, and CBD can inactivate CYP3A4, however it is unknown whether this is clinically significant. A known interaction with dronabinol is ritonavir. Language: en

Abstract 1767: Anti-proliferative and anti-invasive action of omega-3 polyunsaturated fatty acids in stomach cancer cells

Abstract The American Cancer Society has been reported that about 22,220 cases of stomach cancer will be diagnosed and about 10,990 patients will die from this type of cancer in 2014. Omega-3 polyunsaturated fatty acids (ω3-PUFA), including docosahexaenoic acid (DHA) have been suggested to have anti-cancer effects by epidemiological and clinical studies. However, their underlying anti-cancer mechanisms are still unclear in stomach cancer. This study was designed to examine the impact of ω3-PUFA on stomach cancer and the molecular mechanism of their action. DHA reduced cells viability in a dose- and time-dependent manner in human stomach cancer cells including AGS cells. The DHA-induced cells death was in part due to apoptosis, since the cells exhibited increases of apoptotic markers including cleaved PARP and TUNEL-positive cells. DHA also led to autophagic vesicle formation an increase levels of lipidated form LC3B and stimulated the autophagic flux, indicating the involvement of both apoptosis and autophagy in the cytotoxic effect of stomach cancer cells by ω3-PUFA. Moreover, AGS stable cell expressing the ω3-desaturase (fat-1) gene (AGS-fsc) or control vector stable cell (AGS-csc) were eatablished, and the effect of endogenous high level of ω3-PUFA was investigated in AGS cells. The cell proliferation, invasion and colony-formation were inhibited in AGS-fsc compared to AGS-csc. Taken together, these results indicate that ω3-PUFA induce anti-cancer action is related to anti-proliferation and anti-invasive action in stomach cancer cells. Thus, utilization of ω3-PUFA may represent a promising therapeutic approach for chemoprevention and treatment of human stomach cancer. [This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2014R1A1A2008915) and by the Korea government(MSIP) (No. 2007-0054932)] Citation Format: Soyeon Shin, Soyeon Jeong, Soyeon Kim, Jun-Young Heo, Gi-Ryang Kweon, Seung-Kiel Park, Jong-Il Park, Kyu Lim. Anti-proliferative and anti-invasive action of omega-3 polyunsaturated fatty acids in stomach cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1767. doi:10.1158/1538-7445.AM2015-1767

Autotaxin and LPA1 and LPA5 receptors exert disparate functions in tumor cells versus the host tissue microenvironment in melanoma invasion and metastasis.

Autotaxin (ENPP2/ATX) and lysophosphatidic acid (LPA) receptors represent two key players in regulating cancer progression. The present study sought to understand the mechanistic role of LPA G protein-coupled receptors (GPCR), not only in the tumor cells but also in stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express LPA5 and LPA2 receptors but lack LPA1. LPA dose dependently inhibited invasion of cells across a Matrigel layer. RNAi-mediated knockdown of LPA5 relieved the inhibitory effect of LPA on invasion without affecting basal invasion. This suggests that LPA5 exerts an anti-invasive action in melanoma cells in response to LPA. In addition, both siRNA-mediated knockdown and pharmacologic inhibition of LPA2 reduced the basal rate invasion. Unexpectedly, when probing the role of this GPCR in host tissues, it was found that the incidence of melanoma-derived lung metastasis was greatly reduced in LPA5 knockout (KO) mice compared with wild-type (WT) mice. LPA1-KO but not LPA2-KO mice also showed diminished melanoma-derived lung metastasis, suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of LPA1-KO and LPA5-KO animals was apparent 24 hours after injection. However, KO of LPA1, LPA2, or LPA5 did not affect the subcutaneous growth of melanoma tumors. These findings suggest that tumor and stromal LPA receptors, in particular LPA1 and LPA5, play different roles in invasion and the seeding of metastasis.

LRP1-dependent pepsin clearance induced by 2′-hydroxycinnamaldehyde attenuates breast cancer cell invasion

2′-Hydroxycinnamaldehyde inhibits breast cancer cell invasion. This study examined whether 2′-hydroxycinnamaldehyde, acting as a Michael acceptor, interferes with the ligand binding of low-density lipoprotein receptor-related protein 1 to mediate breast cancer cell invasion. Low-density lipoprotein receptor-related protein 1, one of the direct molecular targets of 2′-hydroxycinnamaldehyde, is a multifunctional endocytic receptor. Changes in the thiol oxidation status of cell surface receptor proteins may function as a molecular switch, influencing ligand(s) binding. The oxidation status of extracellular cysteine thiol groups in MCF-7 and MDA-MB-231 cells was examined using a fluorescence-activated cell sorter with thiol-specific fluorescent probes; Matrigel invasion and wound-healing assays were performed to determine the effects of 2′-hydroxycinnamaldehyde on in vitro cell migration. The molecular mechanisms by which 2′-hydroxycinnamaldehyde acts were evaluated by transient knockdown using siRNA or inhibition of low-density lipoprotein receptor-related protein 1 by receptor-associated protein treatment. 2′-Hydroxycinnamaldehyde increased α-2-macroglobulin binding to low-density lipoprotein receptor-related protein 1, which was alleviated by pretreatment of cells with N-acetylcystein. 2′-Hydroxycinnamaldehyde decreased the extracellular pepsin concentration significantly in a low-density lipoprotein receptor-related protein 1- and α-2-macroglobulin-dependent manner. The anti-invasive effect of 2′-hydroxycinnamaldehyde was mitigated with receptor-associated protein pretreatment, suggesting that low-density lipoprotein receptor-related protein 1 is essential for the effects of 2′-hydroxycinnamaldehyde. From these data, we suggest that 2′-hydroxycinnamaldehyde increases the cysteine thiol oxidation status of low-density lipoprotein receptor-related protein 1 extracellular domains, which results in α-2-macroglobulin ligand binding stimulation. Therefore, pepsin clearance in a low-density lipoprotein receptor-related protein 1-α-2-macroglobulin-dependent manner might be an important molecular mechanism in 2′-hydroxycinnamaldehyde exerting its anti-invasive action on breast cancer cells. Furthermore, our data may provide an opportunity to promote the importance of the thiol oxidation status of cell surface receptor proteins for regulating cellular signaling pathways that are important in cancer progression.

MicroRNAs 424 and 503 are mediators of the anti-proliferative and anti-invasive action of the thyroid hormone receptor beta.

The thyroid hormone receptors (TRs) mediate tumor suppressive effects in hepatocarcinoma and breast cancer cells. Here we show that incubation of hepatocarcinoma SK-hep1 cells expressing TRb with the thyroid hormone T3 induces transcription of the polycistronic message coding for microRNAs 424 and 503. TRb binds to the promoter region of these miRNAs and T3 induces an exchange of corepressors and coactivators inducing histone acetylation and transcriptional stimulation. We have validated cell cycle components as targets of these miRNAs. Overexpression of miR-424 mimicked the repressive effect of T3 on cell proliferation, growth in suspension, migration and invasion. Knockdown of miR-424 or miR-503 reduced the inhibitory effect of the hormone. T3 increased miR-424 and miR-503 in breast cancer cells expressing TRb, and this induction is also involved in the anti-invasive effects of the hormone. Furthermore, miR-424 or miR-503 depletion enhanced extravasation to the lungs of hepatocarcinoma cells injected in the tail vein of mice. The levels of these miRNAs were reduced in xenograft tumors formed in hypothyroid nude mice that are more invasive. Therefore, miR-424 or miR-503 mediate anti-proliferative and anti-invasive actions of TRb both in cultured cells and in vivo.

Berberine Hydrochloride Impact on Physiological Processes and Modulation of Twist Levels in Nasopharyngeal Carcinoma CNE-1 Cells

The main purpose of this work was to investigate the effect of berberine hydrochloride (BH) on the proliferation, apoptosis, migration, and invasion of CNE-1 nasopharyngeal carcinoma cells. Our results shed light on the functional components of traditional Chinese herbs for potential use in modern medicine. The CNE-1 cell line was treated with different concentrations of BH and effects on cell viability and proliferation were evaluated using the Cell Counting Kit-8 (CCK-8) assay. Anti-migratory and anti-invasive actions of BH were investigated using wound healing assays and the Millicell Hanging cell culture insert system, respectively. Expression of the epithelial-mesenchymal transition (EMT)-related gene twist (Twist) was analyzed by real-time PCR and Western blotting. Apoptosis was estimated with an annexin-V fluorescein (FITC) apoptosis detection kit, as well as with reference to levels of activated caspase-3 of CNE-1 cells before and after treatment with BH utilizing fluorescence spectroscopy. BH was capable of reducing proliferation and viability of CNE-1 cells in a dose- and time-dependent manner, also demonstrating anti-migratory and anti-invasive capacities which correlated with reduction in expression of Twist. Finally, BH was able to induce significant amounts of apoptosis in CNE-1 cells, as demonstrated by an increase in the activity of caspase-3 and in annexin-V staining following treatment. BH extracted from rhizoma coptidis demonstrated an ability to block proliferation, induce apoptosis, and impair the migration and invasion of the CNE-1 cell line Considering these properties, our results suggest that BH could be an important compound for consideration in the treatment of nasopharyngeal carcinoma.

Melatonin antagonizes hypoxia‐mediated glioblastoma cell migration and invasion via inhibition of HIF‐1α

Hypoxia is a crucial factor in tumor aggressiveness and resistance to therapy, especially in glioblastoma. Our previous results have shown that melatonin exerts antimigratory and anti-invasive action in glioblastoma cells under normoxia. However, the effect of melatonin on migration and invasion of glioblastoma cells under hypoxic condition remains poorly understood. Here, we show that melatonin strongly reduced hypoxia-mediated invasion and migration of U251 and U87 glioblastoma cells. In addition, we found that melatonin significantly blocked HIF-1α protein expression and suppressed the expression of downstream target genes, matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF). Furthermore, melatonin destabilized hypoxia-induced HIF-1α protein via its antioxidant activity against ROS produced by glioblastoma cells in response to hypoxia. Along with this, HIF-1α silencing by small interfering RNA markedly inhibited glioblastoma cell migration and invasion, and this appeared to be associated with MMP-2 and VEGF under hypoxia. Taken together, our findings suggest that melatonin suppresses hypoxia-induced glioblastoma cell migration and invasion via inhibition of HIF-1α. Considering the fact that overexpression of the HIF-1α protein is often detected in glioblastoma multiforme, melatonin may prove to be a potent therapeutic agent for this tumor.

Abstract 4207: CCN5/WISP-2 upregulates p27Kip1 in breast cancer cell lines through AKT/FOXO3a signaling

Abstract CCN5 is a member of the CCN (cysteine rich 61-connective tissue growth factor-nephroblastoma-overexpressed) family of growth factors. Our studies have shown that CCN5 is mainly expressed in less aggressive human breast cancer cells (i.e., MCF-7 and ZR-75-1) and noninvasive lesion (i.e., atypical ductal hyperplasia and ductal carcinoma in situ), whereas its expression is minimally detected in moderately aggressive breast cancer cell lines (i.e., SKBR-3), and it is undetected in triple negative highly aggressive breast cancer cell line (i.e., MDA-MB-231) and invasive lesion. Ectopic expression of CCN5 has been shown to inhibit tumorigenicity in MDA-MB-231-xenograft models therefore suggesting that CCN5 is an anti-invasive molecule. The objective of the present study was to determine how CCN5 overexpression affects cell-cycle progression of breast cancer cells. We found knocking down of CCN5 in non-invasive breast cancer cells significantly downregulated the expression of p27Kip1, a tumor suppressor molecule. In contrast, enforced/ectopic expression of CCN5 overexpression in MDA-MB-231 breast cancer cells caused cell cycle arrest at G1 phase and dramatically reduced proliferation of these cells. CCN5 overexpression results in increased the stability as well as the expression of p27Kip1 protein. The regulation of p27Kip1 by CCN5 is mediated through AKT/FOXO3a signaling. Collectively, the studies suggest that CCN5 exerts its anti-invasive action through the regulation of p27Kip1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4207. doi:1538-7445.AM2012-4207

Antiproliferative and Anti-Invasive Effect of Piceatannol, a Polyphenol Present in Grapes and Wine, against Hepatoma AH109A Cells

Piceatannol is a stilbenoid, a metabolite of resveratrol found in red wine. Piceatannol and sera from rats orally given piceatannol were found to dose-dependently suppress both the proliferation and invasion of AH109A hepatoma cells in culture. Its antiproliferative effect was based on cell cycle arrest at lower concentration (25~50 μM) and on apoptosis induction at higher concentration (100 μM). Piceatannol suppressed reactive oxygen species-potentiated invasive capacity by scavenging the intracellular reactive oxygen species. These results suggest that piceatannol, unlike resveratrol, has a potential to suppress the hepatoma proliferation by inducing cell cycle arrest and apoptosis induction. They also suggest that the antioxidative property of piceatannol, like resveratrol, may be involved in its anti-invasive action. Subsequently, piceatannol was found to suppress the growth of solid tumor and metastasis in hepatoma-bearing rats. Thus, piceatannol may be a useful anticancer natural product.