5FUR Cells Research Articles

Increased soluble E‑cadherin of spheroid formation supplemented with fetal bovine serum in colorectal cancer cells.

Cancer stem cells (CSCs) are known to be a major cause of metastasis, resistance and recurrence. Spheroid formation is one of the methods used to recruit CSCs utilizing an anchorage-independent environment in vitro. It was aimed to investigate the availability of spheroid formation culture methods in the research field of CSCs and resistance using 5-fluorouracil (5-FU)-resistant colorectal cancer cells. The wild type SNU-C5 and 5-FU-resistant SNU-C5 (SNU-C5/5-FUR) cells were cultured as usual (monolayer), and in 3-dimensional non-adhesive environments supplemented with fetal bovine serum (FBS) or growth factors, respectively. The characteristics of the spheroids were evaluated by morphometry, cell viability assay, western blotting, immunocytochemistry and enzyme-linked immunosorbent assay. Spheroid formation was induced in an environment supplemented with FBS, while SNU-C5/5-FUR cells only formed spheres in media supplemented with GFs. Sphere-formed cells showed slower cell proliferation than cells from monolayer, which coincided with an increased level of p21 and a decreased level of β-catenin. Markers for CSCs and drug resistance were not significantly changed after spheroid formation. Sphere-formed cells showed significantly increased levels of soluble E-cadherin, particularly in the environment supplemented with FBS. These results suggested that spheroid formation may be related to soluble E-cadherin, but is not related to CSCs or resistance markers.

DYRK2 downregulation in colorectal cancer leads to epithelial–mesenchymal transition induction and chemoresistance

Colorectal cancer (CRC) is among the most prominent causes of cancer-associated mortality in the world, with chemoresistance representing one of the leading causes of treatment failure. However, the mechanisms governing such chemoresistance remain incompletely understood. In this study, the role of DYRK2 as a mediator of CRC cell drug resistance and the associated molecular mechanisms were assessed by evaluating human tumor tissue samples, CRC cell lines, and animal model systems. Initial analyses of The Cancer Genome Atlas database and clinical tissue microarrays revealed significant DYRK2 downregulation in CRC in a manner correlated with poor prognosis. We further generated LoVo CRC cells that were resistant to the chemotherapeutic drug 5-FU, and found that such chemoresistance was associated with the downregulation of DYRK2 and a more aggressive mesenchymal phenotype. When DYRK2 was overexpressed in these cells, their proliferative, migratory, and invasive activities were reduced and they were more prone to apoptotic death. DYRK2 overexpression was also associated with enhanced chemosensitivity and the inhibition of epithelial–mesenchymal transition (EMT) induction in these LoVo 5-FUR cells. Co-immunoprecipitation assays revealed that DYRK2 bound to Twist and promoted its proteasomal degradation. In vivo studies further confirmed that the overexpression of DYRK2 inhibited human CRC xenograft tumor growth with concomitant Twist downregulation. Overall, these results thus highlight DYRK2 as a promising therapeutic target in CRC worthy of further investigation.

Long-term resistance to 5-fluorouracil promotes epithelial–mesenchymal transition, apoptosis evasion, autophagy, and reduced proliferation rate in colon cancer cells

The drug, 5-fluorouracil (5FU) is a standard first-line treatment for colorectal cancer (CRC) patients. However, drug resistance acquisition remains an important challenge for effective clinical outcomes. Here, we established a long-term drug-resistant CRC model and explored the cellular events underlying 5FU resistance. We showed that 5FU-treated cells (HCT-116 5FUR) using a prolonged treatment protocol were significantly more resistant than parental cells. Likewise, cell viability and IC50 values were also observed to increase in HCT-116 5FUR cells when treated with increasing doses of oxaliplatin, indicating a cross-resistance mechanism to other cytotoxic agents. Moreover, HCT-116 5FUR cells exhibited metabolic and molecular changes, as evidenced by increased thymidylate synthase levels and upregulated mRNA levels of ABCB1. HCT-116 5FUR cells were able to overcome S phase arrest and evade apoptosis, as well as activate autophagy, as indicated by increased LC3B levels. Cells treated with low and high doses displayed epithelial–mesenchymal transition (EMT) features, as observed by decreased E-cadherin and claudin-3 levels, increased vimentin protein levels, and increased SLUG, ZEB2 and TWIST1 mRNA levels. Furthermore, HCT-116 5FUR cells displayed enhanced migration and invasion capabilities. Interestingly, we found that the 5FU drug-resistance gene signature is positively associated with the mesenchymal signature in CRC samples, and that ABCB1 and ZEB2 co-expressed at high levels could predict poor outcomes in CRC patients. Overall, the 5FU long-term drug-resistance model established here induced various cellular events, and highlighted the importance of further efforts to identify promising targets involved in more than one cellular event to successfully overcome drug-resistance.

The mechanism underlying resistance to 5-fluorouracil and its reversal by the inhibition of thymidine phosphorylase in breast cancer cells.

Fluoro-deoxyuridine monophosphate (FdUMP) is an active metabolite of 5-fluorouracil (5-FU) synthesized through two hypothesized pathways: The orotate phosphoribosyl transferase-ribonucleotide reductase (OPRT-RR) pathway and the thymidine phosphorylase-thymidine kinase (TP-TK) pathway. In the present study, the mechanism underlying 5-FU resistance was investigated, focusing on changes in 5-FU metabolism using MCF-7, 5-FU-resistant MCF-7/5-FUR, MDA-MB-231 and 5-FU-resistant MDA-MB-231/5-FUR breast cancer cells. The amount of FdUMP present following treatment with 5-FU was determined by the density of the upper band of thymidylate synthase detected by western blotting, and its changes were investigated. MCF-7/5-FUR cells exhibited 5-FU resistance (36.6-fold), and showed decreased OPRT (-69.3%) and TK (-42.6%) levels. MDA-MB-231/5-FUR cells also exhibited 5-FU resistance (15.8-fold), and showed decreased TP (-79.0%) and increased TK (+184%) levels. MCF-7/5-FUR and MDA-MB-231/5-FUR cells both showed decreased synthesis of FdUMP by 91 and 86%, respectively. In MCF-7 and MCF-7/5-FUR cells, the synthesis of FdUMP was decreased when 5-FU was combined with an RR inhibitor, indicating that FdUMP was synthesized through the OPRT-RR pathway. The synthesis of FdUMP was decreased when 5-FU was combined with a TP inhibitor in MDA-MB-231 cells and combined with an RR inhibitor in MDA-MB-231/5-FUR cells, indicating that the synthesis pathway of FdUMP was changed from the TP-TK pathway to the OPRT-RR pathway on acquiring resistance to 5-FU. Notably, the synthesis of FdUMP was increased and the resistance to 5-FU was reversed in MCF-7/5-FUR cells (half maximal inhibitory concentration (IC50): 219.9 to 0.093 µM) and MDA-MB-231/5-FUR cells (IC50: 157.3 to 31.0 µM) when 5-FU was combined with a TP inhibitor. In conclusion, the metabolism of 5-FU and the mechanism underlying the resistance to 5-FU differed among cell lines, and inhibition of TP reversed resistance to 5-FU, thus suggesting that the combination of 5-FU and a TP inhibitor may be considered a promising cancer therapy.

The Endoplasmic Reticulum Stress Response Mediates Shikonin-Induced Apoptosis of 5-Fluorouracil-Resistant Colorectal Cancer Cells.

Resistance to chemotherapeutic drugs is a significant problem in the treatment of colorectal cancer, resulting in low response rates and decreased survival. Recent studies have shown that shikonin, a naphthoquinone derivative, promotes apoptosis in colon cancer cells and cisplatin-resistant ovarian cells, raising the possibility that this compound may be effective in drug-resistant colorectal cancer. The aim of this study was to characterize the molecular mechanisms underpinning shikonin-induced apoptosis, with a focus on endoplasmic reticulum (ER) stress, in a 5-fluorouracil–resistant colorectal cancer cell line, SNU-C5/5-FUR. Our results showed that shikonin significantly increased the proportion of sub-G1 cells and DNA fragmentation and that shikonin-induced apoptosis is mediated by mitochondrial Ca2+ accumulation. Shikonin treatment also increased the expression of ER-related proteins, such as glucose regulatory protein 78 (GRP78), phospho-protein kinase RNA-like ER kinase (PERK), phospho-eukaryotic initiation factor 2 (eIF2α), phospho-phosphoinositol-requiring protein-1 (IRE1), spliced X-box–binding protein-1 (XBP-1), cleaved caspase-12, and C/EBP-homologous protein (CHOP). In addition, siRNA-mediated knockdown of CHOP attenuated shikonin-induced apoptosis, as did the ER stress inhibitor TUDCA. These data suggest that ER stress is a key factor mediating the cytotoxic effect of shikonin in SNU-C5/5-FUR cells. Our findings provide an evidence for a mechanism in which ER stress leads to apoptosis in shikonin-treated SNU-C5/5-FUR cells. Our study provides evidence to support further investigations on shikonin as a therapeutic option for 5-fluorouracil–resistant colorectal cancer.

Anti-cancer effects of the aqueous extract of Orostachys japonica A. Berger on 5-fluorouracil-resistant colorectal cancer via MAPK signalling pathways in vitro and in vivo

Ethnopharmacological relevanceOrostachys japonica A. Berger, also known as Wa-song in Korea, has traditionally been used as a folk medicine, but the potential anti-cancer effects of aqueous extract of Orostachys japonica (OJe) have not yet been thoroughly investigated. Aim of the studyTo evaluate the anti-cancer effects of OJe, its possible mechanisms of action were investigated in 5-fluorouracil (5-FU) resistant SNU-C5/5-FUR colorectal cancer cells. Materials and methodsThe functional compounds of OJe were identified with high performance liquid chromatography. The anti-cancer effects of OJe in SNU-C5/5-FUR cells were investigated by a cell viability assays, flow cytometry analysis, and a subcutaneous xenograft model employing BALB/c-nude mice. Possible signalling pathways were assayed with Western blotting. ResultsOJe (250 μg/ml) showed anti-cancer effects in SNU-C5/5-FUR cells, that were mediated via apoptosis as well as cell cycle arrest at the G0/G1 phase. Gallic acid and (−)-epicatechin, the major functional components of OJe, induced cell cycle arrest. OJe treatment (250 mg/kg, p.o.) produced a significant anti-proliferative effect in the xenograft model via decreased β-catenin/GSK3β and increased p27 expression. OJe treatment significantly activated ERK and p38 both in vitro and in vivo. ConclusionsThese results suggest that OJe has anti-proliferative effects on 5-FU-resistant colorectal cancer cells via regulation of MAPK signalling pathways.

Abstract 1039: Andrographis enhances 5-fluorouracil-induced anti-tumor effect in colorectal cancer through inhibition of DKK1

Abstract Background: Colorectal cancer (CRC) ranks as the third leading cause of cancer-related deaths in the US. In this malignancy, 5-fluorouracil (5FU)-based treatment remains the mainstay as first-line chemotherapy. Unfortunately, ~50-60% of patients treated with 5FU eventually develop resistance, leading to increased mortality. Hence, a better understanding of the molecular mechanisms underlying 5FU-resistance will lead to development of more effective therapeutic strategies. Our previous studies have shown that, andrographis, a botanical with diverse biological activities, synergistically enhanced 5FU-induced pro-apoptotic effects by mediating Wnt/β-catenin pathways. However, a more comprehensive knowledge of andrographis-mediated synergism with 5-FU remains largely unclear. Methods: We first established 5FU resistant isogenic cell lines of HCT116 and SW480 CRC cells (HCT116 5FUR and SW480 5FUR). To determine the synergistic anti-tumor effects of andrographis and 5FU, we performed cell viability, proliferation and colony formation assays by treating parental and 5FUR cells with andrographis, 5FU and their combination. To explore the molecular mechanisms responsible for the synergistic anti-tumor efficacy, we next performed gene expression microarrays to identify candidate pathways and genes that mediated anti-tumor efficacy of andrographis in 5FUR cells. We analyzed an expression profiling dataset from CRC patients (GSE52735) who either responded or did not respond to 5FU. We also studied the andrographis-mediated synergism with 5FU in a xenograft animal model, and in 3D patient-derived tumor organoids. Results: Combined treatment with andrographis and 5FU exhibited significantly superior effects on cell viability (P<0.01), proliferation (P<0.001) and colony formation capacity (P<0.001), compared to treatment with individual treatments. In line with our in vitro findings, the combined treatment also significantly reduced tumor growth in mice xenografts (P<0.0001) as well as patient-derived tumoroids (P<0.001). By analyzing gene expression profiling data from cell lines and CRC patients, we identified that overexpression of DKK1 gene was a critical event for 5FU resistance in CRC. Moreover, qRT-PCR and western blotting data revealed that andrographis resulted in downregulation of 5FU-induced DKK1 overexpression accompanied with the synergistic anti-tumor effects in animal xenografts (P<0.001) and patient-derived tumor organoids (P<0.001), which were reversed following siRNA-mediated silencing of DKK1 (P<0.01). Conclusions: Our data provide novel evidence for andrographis-mediated anti-tumor synergism with 5-FU through DKK1, highlighting that in addition to its preventive efficacy, andrographis may serve as a potential adjunctive treatment to conventional chemotherapeutic drugs in patients with CRC. Citation Format: Yinghui Zhao, Chuanxin Wang, Ajay Goel. Andrographis enhances 5-fluorouracil-induced anti-tumor effect in colorectal cancer through inhibition of DKK1 [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 1039.

Andrographis overcomes 5-fluorouracil-associated chemoresistance through inhibition of DKK1 in colorectal cancer.

Colorectal cancer (CRC) ranks as the third leading cause of cancer-related deaths in the USA. 5-Fluorouracil (5FU)-based chemotherapeutic drug remains a mainstay of CRC treatment. Unfortunately, ~50-60% of patients eventually develop resistance to 5FU, leading to poor survival outcomes. Our previous work revealed that andrographis enhanced 5FU-induced anti-cancer activity, but the underlying mechanistic understanding largely remains unclear. In this study, we first established 5FU-resistant (5FUR) CRC cells and observed that combined treatment with andrographis-5FU in 5FUR cells exhibited superior effect on cell viability, proliferation, and colony formation capacity compared with individual treatments (P < 0.001). To identify key genes and pathways responsible for 5FU resistance, we analyzed genome-wide transcriptomic profiling data from CRC patients who either responded or did not respond to 5FU. Among a panel of differentially expressed genes, Dickkopf-1 (DKK1) overexpression was a critical event for 5FU resistance. Moreover, andrographis significantly downregulated 5FU-induced DKK1 overexpression, accompanied with enhanced anti-tumor effects by abrogating downstream Akt-phosphorylation. In line with in vitro findings, andrographis enhanced 5FU-induced anti-cancer activity in mice xenografts and patient-derived tumoroids (P < 0.01). In conclusion, our data provide novel evidence for andrographis-mediated reversal of 5FU resistance, highlighting its potential role as an adjunct to conventional chemotherapy in CRC.

Novel quinazolinone MJ‑33 induces AKT/mTOR‑mediated autophagy‑associated apoptosis in 5FU‑resistant colorectal cancer cells.

Novel quinazolinone compounds have been studied in the field of drug discovery for a long time. Among their broad range of pharmacological effects, certain compounds effectively inhibit cancer cell proliferation. MJ-33 is a quinazolinone derivative with proposed anticancer activities that was synthesized in our laboratory. The present study aimed to evaluate the anticancer activity of MJ-33 in fluorouracil (5FU)-resistant colorectal cancer cells (HT-29/5FUR) and to investigate the underlying molecular mechanisms. The cell viability assay results indicated that HT-29/5FUR cell viability was inhibited by MJ-33 treatment in a concentration-dependent manner compared with the control group. The cellular morphological alterations observed following MJ-33 treatment indicated the occurrence of apoptosis and autophagy, as well as inhibition of cell proliferation in a time-dependent manner compared with the control group. The acridine orange, LysoTracker Red and LC3-green fluorescent protein staining results indicated that MJ-33 treatment significantly induced autophagy compared with the control group. The DAPI/TUNEL dual staining results demonstrated increased nuclear fragmentation and condensation following MJ-33 treatment compared with the control group. The Annexin V apoptosis assay and image cytometry analysis results demonstrated a significant increase in apoptotic cells following MJ-33 treatment compared with the control group. The western blotting results demonstrated markedly decreased Bcl-2, phosphorylated (p)-BAD, pro-caspase-9 and pro-caspase-3 expression levels, and notably increased cytochrome c and apoptotic peptidase activating factor 1 expression levels following MJ-33 treatment compared with the control group. Moreover, the expression levels of autophagy-related proteins, including autophagy related (ATG)-5, ATG-7, ATG-12, ATG-16, p62 and LC3-II, were increased following MJ-33 treatment compared with the control group. Furthermore, MJ-33-treated HT-29/5FUR cells displayed decreased expression levels of p-AKT and p-mTOR compared with control cells. The results suggested that MJ-33-induced apoptosis was mediated by AKT signaling, and subsequently modulated via the mitochondria-dependent signaling pathway. Therefore, the results suggested that suppression of AKT/mTOR activity triggered autophagy in the HT-29/5FUR cell line. In summary, the results indicated that MJ-33 inhibited HT-29/5FUR cell viability, and induced apoptosis and autophagy via the AKT/mTOR signaling pathway. The present study may provide novel insight into the anticancer effects and mechanisms underlying MJ-33 in 5FU-resistant colorectal cancer cells.

Lactobacillus-derived metabolites enhance the antitumor activity of 5-FU and inhibit metastatic behavior in 5-FU-resistant colorectal cancer cells by regulating claudin-1 expression.

Lactobacillus plantarum-derived metabolites (LDMs) increase drug sensitivity to 5-FU and antimetastatic effects in 5-FU-resistant colorectal cancer cells (HCT-116/5FUR). In this study, we evaluated the effects of LDMs on the regulation of genes and proteins involved in HCT-116/5-FUR cell proliferation and metastasis. HCT-116/5-FUR cells showed high metastatic potential, significantly reduced tight junction (TJ) integrity, including increased migration and paracellular permeability, and upregulation of claudin-1 (CLDN-1). The genetic silencing of CLDN-1 increased the sensitivity of HCT-116/5FUR to 5-FU and inhibited its metastatic potential by regulating the expression of epithelial-mesenchymal transition (EMT) related genes. Co-treatment of HCT-116/5FUR with LDMs and 5-FU suppressed chemoresistant and metastatic behavior by downregulating CLDN-1 expression. Finally, we designed LDMs-based therapeutic strategies to treatment for metastatic 5-FU-resistant colorectal cancer cells. These results suggested that LDMs and 5-FU cotreatments can synergistically target 5-FU-resistant cells, making it a candidate strategy to overcome 5-FU chemoresistance improve anticancer drug efficacy.

Andrographis-mediated chemosensitization through activation of ferroptosis and suppression of β-catenin/Wnt-signaling pathways in colorectal cancer.

Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality in the USA. As much as 50-60% of CRC patients develop resistance to 5-fluorouracil (5FU)-based chemotherapeutic regimens, attributing the increased overall morbidity and mortality. In view of the growing evidence that active principles in various naturally occurring botanicals can facilitate chemosensitization in cancer cells, herein, we undertook a comprehensive effort in interrogating the activity of one such botanical-andrographis-by analyzing its activity in CRC cell lines [both sensitive and 5FU resistant (5FUR)], a xenograft animal model and patient-derived tumor organoids. We observed that combined treatment with andrographis was synergistic and resulted in a significant and dose-dependent increase in the efficacy of 5FU in HCT116 and SW480 5FUR cells (P < 0.05), reduced clonogenic formation (P < 0.01) and increased rates of caspase-9-mediated apoptosis (P < 0.05). The genomewide expression analysis in cell lines led us to uncover that activation of ferroptosis and suppression of β-catenin/Wnt-signaling pathways were the key mediators for the anti-cancer and chemosensitizing effects of andrographis. Subsequently, we validated our findings in a xenograft animal model, as well as two independent CRC patient-derived organoids-which confirmed that combined treatment with andrographis was significantly more effective than 5FU and andrographis alone and that these effects were in part orchestrated through dysregulated expression of key genes (including HMOX1, GCLC, GCLM and TCF7L2) within the ferroptosis and Wnt-signaling pathways. Collectively, our data highlight that andrographis might offer a safe and inexpensive adjunctive therapeutic option in the management of CRC patients.

Abstract 571: Andrographolide enhances sensitivity to 5-fluorouracil by targeting ferroptosis-related genes in colorectal cancer

Abstract Purpose: Despite improvements in response rates from various treatment strategies, the 5-year survival rates for metastatic colorectal cancer (mCRC) remain dismal (~10-15%). One of the primary reasons for CRC therapy failure is development of acquired drug resistance to first line chemotherapies such as 5-fluorouracil (5FU), and nearly half of all mCRC patients are resistant to 5FU-based therapies. These limitations highlight the need for novel treatment strategies that offer improved efficacy and can help overcome acquired chemoresistance and enhance anti-cancer efficacy of conventional chemotherapies. Considering that some naturally-occurring botanicals have chemo-sensitizing properties, we analyzed the potential of andrographolide, a botanical with key biological activities, including anti-inflammatory, antitumorigenic, apoptotic, antiangiogenic and immunomodulatory activities, in reversing chemoresistance in CRC, and also delineated the associated molecular pathways underpinning this activity. Experimental design: We performed a series of in-vitro and in-vivo experiments to investigate the chemo-sensitizing ability of andrographolide in 5-FU resistant (5FUR) CRC cells, xenografts animal models and CRC-derived organoids. Furthermore, we performed gene expression microarrays and other mechanistic studies to explore the molecular mechanisms responsible for its chemo-sensitizing activity. Results: Andrographolide treatment significantly enhanced the efficacy of 5FU in HCT116 5FUR and SW480 5FUR cells, as evidenced by enhanced apoptosis and decreased proliferation rates (p&amp;lt;0.05). In line with our in-vitro findings, treatment with andrographolide also led to significant tumor growth inhibition in a xenograft animal model (p&amp;lt;0.0001). Likewise, in accordance with the results from cell lines and mice xenografts, the combination of 5FU and andrographolide significantly decreased the growth of patient-derived tumor organoids compared to those treated with 5FU alone (p&amp;lt;0.01 for patient 1, p&amp;lt;0.05 for patient 2). Through microarray-based gene expression profiling analysis in 5FU-resistant CRC cells, we identified ferroptosis and WNT signaling as the key cancer associated pathways responsible for andrographolide mediated chemo-sensitization in CRC cells. Taken together, using genome-wide gene expression analysis, we delineated the mechanism underlying the chemo-sensitizing properties of andrographolide and made a case for the potential use of andrographolide for enhancing the therapeutic efficacy of 5FU in CRC. Conclusion: Our data provides first evidence for andrographolide mediated sensitization to 5FU based therapy, through modulation of ferroptosis pathway in CRC. These results highlight the potential adjunctive therapeutic role for andrographolide in combination with conventional chemotherapeutic drug in patients with CRC. Citation Format: Priyanka Sharma, Tadanobu Shimura, Jasjit K Banwait, Ajay Goel. Andrographolide enhances sensitivity to 5-fluorouracil by targeting ferroptosis-related genes in colorectal 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 571.

PO-472 Chemotherapy resistance-associated epithelial to endothelial transition in gastric cancer

Introduction Gastric cancer (GC) is the fifth most common cancer worldwide and the third leading cause of cancer-related deaths. To date, gastrectomy and chemotherapy are the only therapeutic options, but drug resistance is the main cause for treatment failure. Vasculogenic mimicry (VM) is a new model of neovascularization in aggressive tumours and has been correlated with poor prognosis in GC patients. Our group has developed chemotherapy-resistant GC cells using the Caucasian adenocarcinoma cell line AGS and three drugs among the most used in clinic (5-fluorouracil, cisplatin and paclitaxel) henceforward denominated 5FUr, CISr, TAXr. Our study has highlighted phenotypical differences among chemo-sensitive and chemo-resistant cell lines such as acquisition of stem-like phenotype and increased capacity to form vessels. Material and methods Establishment of AGS resistant cell lines exposing cells to increasing dilution of drugs for over 9 months up to dilutions higher than IC50 values initially verified on AGS cells through MTT analysis. Quantitative RT-PCR, flow cytometry and western blot analysis for stemness and VM markers. Vasculogenic mimicryassay Results and discussions AGS cells acquired chemoresistance as indicated by the increase of IC50 values in drug-treated cells with respect to AGS. Furthermore, MTT assay highlighted that there is not cross-resistance among 5FUr, CISr and TAXr. Supportive data is that cells are MDR1 negative. Resistant cells showed an upregulation of Yamanaka factors either in qPCR and flow cytometer analysis, and particularly interesting is ALDH overexpression in 5FUr. TWIST upregulation suggested the investigation of VM which resulted particularly enhanced in 5FUr cells which demonstrated their ability to form and sustain vessels up to 96 hours in the tube formation assay. Markers of VM such Laminin γ2 and Ephrin A2 showed an increase in resistant cells and especially in 5FUr. Conclusion One of the most interesting result is that 5FUr cells acquire stemness properties and are positive to the tube formation assay suggesting that VM might be one mechanisms adopted by cells to avoid drugs exposure. These findings suggest that acquisition of chemoresistance could cause a relapse of disease in which tumour cells take advantage of their capability to perform VM in order to self-sustain their growth and that may be cause of poor outcomes.

5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin

BackgroundDevelopment of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma.Methods5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin.ResultsThe 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS2a, altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105-expressing 5-FUR cells, whereas little effect was seen in the CD105-negative non-resistant parental cancer cell lines. Strikingly, using the intracellular drug delivery method photochemical internalization (PCI) by combining light-controlled activation of the TPCS2a with nanomolar levels of CD105-saporin resulted in strong cytotoxic effects in the 5-FUR cell population.ConclusionOur findings suggested that autophagy is an important resistance mechanism against the chemotherapeutic drug 5-FU in pancreatic cancer cells, and that inhibition of the autophagy process, either by CQ or lysosomal photodamage, can contribute to increased sensitivity to 5-FU. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells in vitro. In conclusion, PCI-based targeting of CD105 may represent a potent anticancer strategy and should be further evaluated in pre-clinical models.

Long noncoding RNA XIST is a prognostic factor in colorectal cancer and inhibits 5-fluorouracil-induced cell cytotoxicity through promoting thymidylate synthase expression.

A major reason for the failure of advanced colorectal cancer (CRC) treatment is the occurrence of chemoresistance to 5-fluorouracil (5FU)-based treatment. Recent studies have shown that long non-coding RNAs (lncRNAs) are critical regulators in chemoresistance. By using the next generation HiSeq sequencing assay, we identified lncRNAs showing differential expression levels in 5FU resistant and non-resistant CRC patients. RT-qPCR was then performed for validation in tissues and serum samples, and lncRNA XIST was verified to be up-regulated in non-responding patients and have considerable diagnostic potential to identify responding patients from non-responding patients. In addition, increased serum XIST level was associated with poor response and lower survival rate in CRC patients receiving 5FU-based treatment. Subsequently, the 5FU resistant (5FU-R) cell lines were established, and lncRNA XIST was significantly up-regulated HT29 5FU-R and HCT116 5FU-R cells. Furthermore, knockdown of XIST reversed 5FU resistance while enhanced XIST could restrained the 5FU-induced cell cytotoxcity in both CRC cell lines. Western blotting and immunofluorescence analysis indicated that XIST promoted the expression of thymidylate synthase, a critical 5FU-targetd enzyme. In conclusion, our integrated approach demonstrates that increased expression of lncRNA XIST3 in CRC confers a potent poor therapeutic efficacy, and that lncRNA XIST participated in 5FU resistance through promoting the expression of thymidylate synthase. Thus, specific silence oflncRNA XIST could be a future direction to develop a novel therapeutic strategy to overcome 5FU resistance of CRC patients.

Epigallocatechin-3-gallate targets cancer stem-like cells and enhances 5-fluorouracil chemosensitivity in colorectal cancer.

Resistance to cytotoxic chemotherapy is a major cause of mortality in colorectal cancer (CRC) patients. A small subset of cancer cells, termed “cancer stem cells” (CSCs), are believed to be key contributors of chemoresistance and tumor recurrence. Recently, epigallocatechin-3-gallate (EGCG), an active catechin present in green tea, has been shown to suppress CSC growth in various cancers, but whether it can specifically target CSCs and subsequently sensitize chemoresistant CRC cells to standard of care chemotherapeutic treatments remains unknown. Herein, we investigated the chemosensitizing effects of EGCG in 5-fluorouracil (5FU)-resistant (5FUR) CRC cells and spheroid-derived CSCs (SDCSCs), and interrogated the underlying molecular mechanisms responsible for its chemopreventive activity. EGCG enhanced 5FU-induced cytotoxicity and inhibited proliferation in 5FUR cell lines through enhancement of apoptosis and cell cycle arrest. The 5FUR cells showed higher spheroid forming capacity compared to parental cells, indicating higher CSC population. EGCG treatment in these cells resulted in suppression of SDCSC formation and enhanced 5FU sensitivity to SDCSCs. Furthermore, EGCG suppressed Notch1, Bmi1, Suz12, and Ezh2, and upregulated self-renewal suppressive-miRNAs, miR-34a, miR-145, and miR-200c, which are some of the key pathways targeted in 5FUR CRC cells. These findings were validated in vivo, wherein EGCG treatment resulted in inhibited tumor growth in a SDCSC xenograft model. Collectively our data provide novel and previously unrecognized evidence for EGCG-induced sensitization to 5FU through targeting of CSCs in CRC. Our data highlight that in addition to its chemopreventive ability, EGCG may serve as an adjunctive treatment to conventional chemotherapeutic drugs in CRC patients.

Curcumin mediates chemosensitization to 5-fluorouracil through miRNA-induced suppression of epithelial-to-mesenchymal transition in chemoresistant colorectal cancer.

Resistance to cytotoxic chemotherapy is a major cause of mortality in colorectal cancer (CRC) patients. Chemoresistance has been linked primarily to a subset of cancer cells undergoing epithelial-mesenchymal transition (EMT). Curcumin, a botanical with antitumorigenic properties, has been shown to enhance sensitivity of cancer cells to chemotherapeutic drugs, but the molecular mechanisms underlying this phenomenon remain unclear. Effects of curcumin and 5-fluorouracil (5FU) individually, and in combination, were examined in parental and 5FU resistant (5FUR) cell lines. We performed a series of growth proliferation and apoptosis assays in 2D and 3D cell cultures. Furthermore, we identified and analyzed the expression pattern of a subset of putative EMT-suppressive microRNAs (miRNAs) and their downstream target genes regulated by curcumin. Chemosensitizing effects of curcumin were validated in a xenograft mouse model. Combined treatment with curcumin and 5FU enhanced cellular apoptosis and inhibited proliferation in both parental and 5FUR cells, whereas 5FU alone was ineffective in 5FUR cells. A group of EMT-suppressive miRNAs were upregulated by curcumin treatment in 5FUR cells. Curcumin suppressed EMT in 5FUR cells by downregulating BMI1, SUZ12 and EZH2 transcripts, key mediators of cancer stemness-related polycomb repressive complex subunits. Using a xenograft and mathematical models, we further demonstrated that curcumin sensitized 5FU to suppress tumor growth. We provide novel mechanistic evidence for curcumin-mediated sensitization to 5FU-related chemoresistance through suppression of EMT in 5FUR cells via upregulation of EMT-suppressive miRNAs. This study highlights the potential therapeutic usefulness of curcumin as an adjunct in patients with chemoresistant advanced CRC.

Dual drug loaded vitamin D3 nanoparticle to target drug resistance in cancer

Overcoming drug resistance is one of the most challenging problems in cancer chemotherapy. Drug cocktails can overcome the drug resistance. However, multiple drug combinations lead to multifold increment of off-target toxicity, as well as the delivery of the required therapeutic amount of combined drugs remains problematic. To address these problems, we have developed a sub 200 nm vitamin D3 nanoparticle, which can contain a rational combination of dual drugs (PI103 and cisplatin or doxorubicin or proflavine). The size, shape and morphology of these dual drug containing vitamin D3 nanoparticles were characterized by DLS, FESEM, AFM and TEM. The nanoparticles released the dual drugs in high quantity at pH = 5.5 compared to pH = 7.4 in a slow and sustained manner over 72 h with stability over 15 days at 37 °C, as well as 4 °C. These dual drug loaded nanoparticles induced increased cell death in human hepatocellular carcinoma, Hep3B cells at 24 h compared to monotherapy; moreover, they were effective against cisplatin-resistant cells (Hep3B-R) as well. VitD3–PI103–CDDP-NP and vitD3–PI103–Dox-NP showed cytotoxicity by inducing apoptosis through DNA damage. Furthermore, vitD3–PI103–CDDP-NP showed considerably improved efficacy in 5-fluorouracil (5-FU) resistant Hep3B–5FU-R cells; its activity was even better compared to 5-FU. Finally, vitD3–PI103–proflavine-NP internalized into Hep3B-R cells considerably faster (within 3 minutes) compared to Hep3B cells, as visualized by fluorescent microscopy. Therefore, these dual drug loaded nanoparticles can successfully overcome the trauma of drug resistance and have the potential to be applied into the clinics for improved cancer therapeutics.

Mo1904 Curcumin Inhibits Epithelial-to-Mesenchymal Transition and Enhances 5-Fluorouracil Sensitivity by Targeting Specific miRNAs in Chemoresistant Colorectal Cancer

Background: Resistance to chemotherapy is one of the major causes of mortality in colorectal cancer (CRC) patients. Chemoresistance has been linked to a subset of ‘cancer cells with stem cell-like' properties undergoing epithelial-to-mesenchymal transition (EMT). Emerging evidence indicates that microRNAs (miRNAs) play a critical role in regulating pathways involved in cancer stemness including polycomb repressive complexes (PRC) and EMT. Curcumin, a potent anti-inflammatory and anti-cancer botanicalmodulates multiple miRNAs, and has been shown to enhance chemosensitivity of chemotherapeutic drugs, such as 5fluorouracil (5FU). However, the molecular mechanisms underlying chemoresistance and the involvement of specific miRNAs and the downstream gene targets mediating EMT and regulation of PRC proteins in CRC remains unclear. Aim: We determined whether curcuminmediated chemosensitization manifests through regulation of specific miRNAs that regulate EMT and polycomb complexes in 5FU-resistant CRC cells. Methods: We first established 5FU resistant (5FUR) CRC cell lines by culturing HCT116 and SW480 cells to increasing concentrations of 5FU for several months. Thereafter, we examined the effects of curcumin (2.5-30 μM) on cellular proliferation, apoptosis, cell-cycle dynamics, and migration in parental and 5FUR cells. Next, we systematically explored the role of curcumin in a subset of putative EMT and PRC-related miRNAs in 5FUR cells. Finally, we validated the chemosensitizing effects of curcumin in a xenograft mice model. Results: Curcumin treatment resulted in inhibited proliferation and migration, increased apoptosis and G2/M arrest in all cell lines, but combined treatment with curcumin and 5FU demonstrated significant synergistic effects in 5FUR cell lines. Interestingly, curcumin upregulated the expression of EMT and PRC related tumor suppressive miR-101, 200b, 200c, 141 and 34ain 5FUR cells. We identified that curcumin suppressed EMT in 5FUR cell lines by down-regulating ZEB1, and inhibited PRC-mediated transcription by targeting EZH2, BMI1 and SUZ12, which dictate cancer Mechanistic investigations revealed that ectopic expression of miR-200c enhanced 5FU sensitivity by down-regulating ZEB1 and BMI1 expression, while siRNA knockdown of miR-200c further enhanced 5FU resistance in 5FUR cells. In addition, we successfully demonstrated this phenomenon in a xenograft model, wherein curcuminmediated 5FU sensitization significantly suppressed tumor growth in these animals. Conclusion: We provide novel mechanistic evidence for curcumin-mediated enhancement of 5FU chemosensitivity through suppression of EMT and PRC in 5FUR cells via coordinated modulation of specific-miRNAs that control cancer stemness. This study highlights the potential therapeutic usefulness of curcumin as a potential adjunct in chemoresistant CRC patients.

Abstract 2276: Proteomic analysis of chemoresistance in colorectal cancer cells: potential paracrine mechanisms of resistance

Abstract Background: Chemoresistance occurs in nearly all patients with metastatic colorectal cancer (CRC), and mechanisms to reverse chemoresistance remain elusive. We tested the hypothesis that CRC cells resistant to 5-fluorouracil (5FU-R) and oxaliplatin (Ox-R) exhibit proteomic profiles that may identify previously unrecognized mediators of resistance. In prior studies, we found that conditioned media from oxaliplatin-resistant HT29 cells (Ox-R) could mediate growth and chemoresistance in chemonaive parental HT29 cells in vitro. We sought to identify soluble factors in conditioned media that are potential mediators of the paracrine cell survival mechanisms. Methods: Parental HT29 (Par) cells were grown in increasing concentrations of 5-FU and oxaliplatin to generate 5FU-R and Ox-R cells. Protein from cell lysates and conditioned media (CM) were analyzed by liquid chromatography-mass spectrometry (LC-MS), and spectral counts were compared. Antibody-conjugated bead technology and ELISAs were used to obtain cytokine profiles of CM. Reverse phase proteomic arrays (RPMA) were used to determine signal transduction pathways activated in cells treated with CM. Ox-R cells were injected into nude mice to determine the paracrine effect on Par cells growing on the opposite flank. Results: Chemoresistant cells displayed significantly different proteomic profiles. In 5FU-R cells, pathways involving oxidative phosphorylation, inositol metabolism, actin cytoskeleton signaling, regulation of actin-based motility by Rho, and ATM signaling were significantly altered vs Par cells. In Ox-R cells, pathways mediating pyruvate metabolism, integrin signaling, caveolar-mediated endocytosis signaling, and mitochondrial dysfunction were among the most altered vs Par cells. Comparison of 5FU-R and Ox-R cells revealed differences in RNA post-transcriptional modification, ERK/MAPK, RAN, and chemokine signaling. Cytokine profiling demonstrated a significant increase in stem cell factor/c-Kit ligand (p=0.02) and a decrease in TRAIL (p=0.008) and IL-10 (p=0.04). RPPA analysis demonstrated early phosphorylation of EGFR and MEK1 followed by GSK, and mTOR activation. Ox-R tumors growing in vivo induced faster and larger tumor growth of contralateral Par tumors indicating a systemic effect. Conclusions: Chemoresistant CRC cells exhibit proteomes that reflect specific survival pathways, with many previously unrecognized potential mediators of resistance. Analysis of soluble factors from chemoresistant CRC cells demonstrates the presence of numerous potential mediators of cancer cell survival that may act, not only in an autocrine/paracrine manner, but also systemically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2276.