Colon Cancer Cell Line WiDr Research Articles

Anticancer activity of Acmella uliginosa against WiDr colon cancer cell line and T47D breast cancer cell line

Cancer is chronic disease that contribute to increasing of death in the world, containing of breast cancer and colon cancer. Natural bioactive compounds have been an important source to exploring drug candidate and drug discovery. Acmella uliginosa is herbaceous plant that has many pharmacology activities. The aim of this study was analysis anti-cancer activity of A. uliginosa. The first step, A. uliginosa was extracted with ethanol and n-hexane, then the extract was evaporated using a rotary evaporator and then analysis to determine phytochemistry compound. Then, brine shrimp lethal test (BSLT) for analysis toxicity effect and preliminary screening anti-cancer compound as well. Moreover, anti-cancer cytotoxic was analyzed using MTT method. This study shows that A. uliginosa contains several phytochemical compounds such as steroid, alkaloid and flavonoid. The preliminary testing of anticancer show n-hexane extract more potent than ethanolic extract, the LC50 value of ethanol and n-Hexane extract was 174.8751mg/L and 60.06807mg/L, respectively. Then the cytotoxic effect of n-hexane extract against WiDr colon cancer cell line and T47D breast cancer cell line was 628.48 µg/ml and 1235.84 µg/ml and 1235.84mcg, respectively. Therefore, n-hexane extract was chosen to be a good candidate for further studies as a new anticancer drug in the treatment of colon cancer.

Overview of wound assay methods extract ethanol leaves soursop (annona muricata) on widr colon cancer cells

Introduction: Soursop leaves have various contents of alkaloid, flavonoid, and acetogenin compounds. Soursop ethanol extract leaves (Annona muricata) showed many beneficialsas antivirus, anti-hyperglycemic, anticancer, etc. As anticancer showed with cytotoxicity insome cancer cell line.But there was no research about wound assay in Widr colon cancercell line as a model of colon cancer diseases. This research carried out to explore theoverviewofwoundassaymethodofsoursopethanolextractleavesininhibitingmigrationofWiDrcoloncancercells. Methods: The series of studies included extra ethanol cytotoxic tests of soursop leaves (Annona muricata L.) on WiDr colon cancer cells usingMTT to assess cell viability and antimigration testing using the scratch wound healingassay method to assess the presentation of closure of WiDr colon cancer cell migration. Results: Cytotoxic test of soursop ethanol extract leaves (Annona muricata L.) on WiDr colon cancer cells obtained IC50 values of 905.77 µg/ml and the cell antimigration test at 24 hours obtained the greatest migration inhibition, namely soursop ethanol extract leaves (Annona muricata L.) at a dose of ½ IC50 or equal to 452.85 µg/ml. Conclusions: Soursop ethanol extract leaves (Annona muricata L.) has an effect on inhibiting migration of WiDr colon cancer cells so that it can be used instead of chemotherapy which can prevent and inhibit the growth of cancer cells.

The cytotoxic and apoptotic effects of wild and polyploidy genotype of Artemisia cina extracts on the WiDr colon and HTB-183 lung cancer cell lines

Abstract. Kristiani EBE, Kasmiyati S, Herawati MM. 2021. The cytotoxic and apoptotic effects of wild and polyploidy genotype of Artemisia cina extracts on the WiDr colon and HTB-183 lung cancer cell lines. Biodiversitas 22: 2844-2852. Artemisia belongs to the Asteraceae family, usually used for traditional treatments of various diseases in China. Artemisia cina Berg ex Poljakov, which lots found in Indonesia, has not been studied much like a cancer drug. The research aims were (i) to compare the bioactive content of hexane and ethyl acetate extract of wild (TWN and KJT) and polyploidy type (J and M) of A. cina and its cytotoxicity on WiDr and HTB-183 cancer cells, (ii) to evaluate the cytotoxicity mechanism of the most substantial extract of it. All of the extracts were prepared by maceration methods using hexane and ethyl acetate separately. The research method used was quantitative with experimental design. The determination of quercetin, kaempferol, and artemisinin used HPLC. The cytotoxicity of the extract determined using the MTT method. The assay of the specific protein related to apoptosis using ICC assay. The content of three bioactive compounds on ethyl acetate extracts higher than hexane extract, in which the extract of J and KJT genotypes had higher levels than M and TWN genotypes. The IC50 value (µg/mL) of extracts against cancer cell lines tested was about 400-700 hexane extracts and 200-500 ethyl acetate extracts. The BCl-2 expressions of both cancer cell lines decreased by treating TWN-EA and M-EA, while the P53 expressions increased. The TWN-EA induced apoptosis of HTB-183 cell lines through caspase-8 and caspase-9 pathways, but M-EA induced caspase-9 pathway only. The TWN and M genotypes were potential to use as anticancer agents on colon and lung cancer.

Exploring anticancer activity of wild and polyploid mutant of Artemisia cina

Abstract. Kasmiyati S, Kristiani EBE, Herawati MM, Rondonuwu FS. 2021. Exploring anticancer activity of wild and polyploid mutant of Artemisia cina. Biodiversitas 22: 1227-1234. The research aims were to explore the anticancer compounds in wild type (W) and polyploid mutant (P) of Artemisia cina Berg ex Poljakov using NIR and compare their cytotoxicity and selectivity on WiDR colon cancer and HTB183 lung cancer cell lines and Vero normal cell line. The W was obtained from B2P2TOOT Tawangmangu, Indonesia while P was by inducing A. cina shoot culture with 100 mg/l colchicine for 48 hours. They were extracted with hexane using the soxhletation method for 6 hours. The anticancer compounds were detected using NIR. The cytotoxic activity was determined using MTT assay, with Doxorubicin (D) as a control. The calculation of IC50 value used SPSS16 with a Probit analysis. The P contained the determined four compounds while the W contained no rutin. The IC50 values of W, P, and D were 295.5, 84.1, and 0.5 µg/mL on WiDr, 322.4, 128.6, and 39.9 µg/mL on HTB 183, whereas on Vero were 104, 315.6, and 295.5 µg/mL respectively. The selectivity indexes of W, P, and D were 34, 4, and 91 on WiDr, while HTB 183 was 31, 3, and 7, respectively. The P contained artemisinin, quercetin, kaemferol, and rutin, while the W contained no rutin. The cytotoxicity of both plants was less than doxorubicin. Both plants were selective on WiDR and HTB 183.

Synergistic anticancer effects of phycocyanin and Citrullus colocynthis extract against WiDr, HCT-15 and HCT-116 colon cancer cell lines

BackgroundWe investigated the effect of C. colocynthis extract and phycocyanin on the growth of colorectal cancer (CRC) cells, and assessed the expression of caspase 3 and Bax encoding genes. MethodsWiDr, HCT-116 and HCT-15 CRC cells were treated with various concentrations of C. colocynthis and phycocyanin extracts for 24, 48 & 72 h. The cell cytotoxicity was measured by MTT assay. Expression of Caspase 3 and Bax genes was evaluated by quantitative real-time PCR (RT-qPCR). ResultsThe combination of extract + phycocyanin as well as C. colocynthis singly unraveled higher inhibitory effects against cancer cells compared to phycocyanin and control. The IC50 values for phycocyanin and the extract + phycocyanin included 6.2 ± 0.86 and 3.8 ± 1.06 μg/μL against ViDr, 6.90 ± 1.11 and 5.87 ± 3.43, respectively. Their cytotoxicity was low against normal cells. The expression of Bax gene was significantly increased in exposure of extract + phycocyanin and also C. colocynthis extract treatment. In addition, the combination of extract + phycocyanin and C. colocynthis extract significantly increased the expression of caspase 3 gene in CRC cell lines. ConclusionC. colocynthis extract, as well as the combination of extract + phycocyanin increased the expression of Bax and caspase 3 genes, indicating intervention in the apoptosis in cancer cells. Although the expression of Bax and Caspase 3 is a major anticancer mechanism of extract and phycocyanin, this finding and other mechanisms require further investigations.

Cytotoxicity and Antiproliferation of Phycocyanin from Spirulina platensis Extract on WiDr Colon Cancer Cell Line

Phycocyanin from Spirulina platensis extract has anticancer activity against various types of cancer cell cultures. However study about its effect on colon cancer cell lines, especially the WiDr, has not been reported before. This study aimed to reveal the anticancer activity of phycocyanin from Spirulina platensis extract on WiDr cells. The research was an in vitro experimental study, with the investigation on cytotoxicity also antiproliferation as the anticancer parameters. Both cytotoxicity and antiproliferation test was conducted through MTT assay to observe the visualization and inhibition of proliferation of different concentrations of phycocyanin in several incubation times on the WiDr colon cancer cell line. The obtained data were then processed statistically with the Two Way ANOVA test at a significance value of p <0.05 and followed with the Post Hoc test since there were significant differences. Based on the results, it could be postulated that phycocyanin extracted from freshwater Spirulina platensis was classified as non-toxic (IC50 of 855 µg/ml). Consequently, it is less potential to be used as the treatment for colon cancer. However, phycocyanin could inhibit the proliferation of the WiDr cell for approximately 47.4%, specifically at the concentration of 1710 µg/ml for 72 hours. It could be concluded that freshwater phycocyanin is less effective as an anticancer substance. The benefit of this study is to provide the new scientific evidence of the contrary results of freshwater phycocyanin activity from Spirulina platensis as an anticancer agent of colon cancer.

Phytochemical analysis and cytotoxic activities of two distinct cultivars of Ganyong rhizomes (Canna indica) against the WiDr colon cancer cell line

Abstract. Ifandari, Widyarini S, Nugroho LH, Pratiwi R. 2020. Phytochemical analysis and cytotoxic activities of two distinct cultivars of Ganyong rhizomes (Canna indica) against the WiDr colon cancer cell line. Biodiversitas 21: 1660-1669. Canna indica L is an edible plant with high nutrient content and positive health benefits. In Indonesia, edible canna is divided into two cultivar groups: red edible canna and green edible canna. Canna indica L. is investigated in this study as a potential medicinal plant for colon cancer in WiDr cell lines. The phytochemical contents of two distinct cultivars of edible canna (known in Indonesia as ganyong), namely red edible canna and green edible canna. These two discrete samples were extracted by using two different solvents, dichloromethane and ethanol, followed by evaluation using Fourier-transform infrared (FTIR) spectroscopy in order to detected functional group. In the analysis of the composition of the elements of chemical compounds, the samples subsequently injected into the liquid chromatography−high-resolution mass spectrometry (LC-HRMS). The cell viability assay was performed to evaluate the cytotoxic activities of extracted compounds from two cultivars of edible canna in two different solvents. FTIR results demonstrated that the red and green edible canna extracts contained 17 and 18 functional group compounds, respectively. Furthermore, the cell viability was lower in WiDr cells treated with dichloromethane extract of red than green edible canna or the ethanol extract of red edible canna. Notably, LC-HRMS results of the most potent dichloromethane extract of red edible canna revealed the presence of fatty acid compounds. These findings indicate that the two cultivars of edible canna could potentially be utilized as promising candidates for colon cancer treatment.

Penentuan Aktivitas Antikanker Fraksi Etil Asetat Daun Bandotan (ageratum conyzoides linn.) Terhadap Cell Line Kanker Kolon WiDr

A research to test the anticancer activity of in vitro anticancer activity against colon cancer cell line WiDr Ageratum conyzoides Linn.leafs. The aims of this study are to determine anticancer activity using MTT method. The method used is the extraction of secondary metabolites using maceration with n-hexane, ethyl acetate and ethanol, followed by TLC. Crude extract maceration results that have the highest yield selected and separated by fractions using KKV. Fractions with the greatest weight was chosen to be tested anticancer activity against WiDr cell line by MTT assay. Phytochemical screening and identification of compounds by GC-MS carried out on the fraction which has the lowest IC50 value. Maceration results showed that crude ethyl acetate extract had the highest amount of yield at 7.31% and has the best separation results of TLC is characterized by the highest number of stains. Separation by KKV produce 21 fractions, 4 fractions were selected based on the weight of the total of the fraction 6, 13, 14 and 15. Test anticancer activity with a concentration of 62.5; 125; 250; 500 and 1000 mg mL-1 shows the fraction 6 has the lowest IC50 value is 251.48 mg mL-1. Based on this, the fraction 6 does not have anticancer activity. Although, the phytochemical screening showed alkaloids and terpenoids as well as the result of identification with GC-MS indicated the presence of compounds 2H-benzopyran and neophytadiene
 Keywords: Ageratum conyzoides Linn. leaves, Column Cromatography Vacuum, anticancer, phytochemical, GC-MS

Cytotoxicity of Tetrahydropentagamavunon-0 (THPGV)-0 and Tetrahydropentagamavunon-1 (THPGV-1) in Several Cancer Cell Lines

Tetrahydropentagamavunon-0 (THPGV-0) and Tetrahydro-pentagamavunon-1 (THPGV-1), are analogs of a curcumin metabolite, tetrahydrocurcumin, and a derivate of Pentagama-vunon-0 (PGV-0) and Pentagamavunon-1 (PGV-1), respectively. THPGV-0 and THPGV-1 have been successfully synthesized and are investigated for their anticancer potency. Cytotoxic assays were performed toward several cancer cell lines to determine values of the IC 50 against those cell lines. Assessing cytotoxicity in Vero normal cell line showed the selectivity of those compound. THPGV-1 showed highest cytotoxic activity in lymphoma Raji cells, a suspension cell line, with an IC 50 of 180mM. Both THPGV-0 and THPGV-1 showed similar potencies in T47D breast cancer cell line with IC 50 values of 250-270mM. Regardless their high selectivity, however, cytotoxic activities of THPGV-0 and THPGV-1 were lower compared to PGV-0 and PGV-1 in HeLa cervical, T47D breast, and WiDr colon cancer cell lines. Further study using different types of cancer cell lines and confirmation of cell viability by another assays and apoptosis detection may give more benefit.

Cytotoxic Effect of Polyisoprenoids from Rhizophora mucronata and Ceriops tagal Leaves against WiDr Colon Cancer Cell Lines

The mangrove plant is known to produce secondary metabolite compounds, mainly isoprenoids. Polyisoprenoids (dolichol and polyprenol) are known to have antimicrobial, anticancer and antiviral activity. Therefore, this study aimed to determine the cytotoxic effects of polyisoprenoids from Rhizophora mucronata and Ceriops tagal leaves by evaluating the induction of apoptosis and cell cycle arrest and the inhibition of the expression of Bcl-2 and cyclin D1 in WiDr colon cancer cells. Cell death was determined based on IC50 values in MTT assays. The induction of apoptosis and alterations in the cell cycle were observed by flow cytometry. The expression of Bcl-2 and cyclin D1 proteins, which play a role in apoptosis and cell cycle regulation, was observed by immunocytochemistry. The results showed that polyisoprenoids from R. mucronata and C. tagal leaves exhibited toxicity against the WiDr cell line, with IC50 values of 278 ± 5.77 and 276 ± 9.54 μg/mL, respectively. Polyisoprenoids from R. mucronata and C. tagal leaves significantly induced apoptosis and caused cell cycle arrest in G0/G1 phase, while also decreasing the expression of Bcl-2 and cyclin D1. Our results confirmed that polyisoprenoids from R. mucronata and C. tagal leaves have the potential to be developed as anticancer agents for colon cancer.

Modelling signalling networks from perturbation data.

Intracellular signalling is realized by complex signalling networks, which are almost impossible to understand without network models, especially if feedbacks are involved. Modular Response Analysis (MRA) is a convenient modelling method to study signalling networks in various contexts. We developed the software package STASNet (STeady-STate Analysis of Signalling Networks) that provides an augmented and extended version of MRA suited to model signalling networks from incomplete perturbation schemes and multi-perturbation data. Using data from the Dialogue on Reverse Engineering Assessment and Methods challenge, we show that predictions from STASNet models are among the top-performing methods. We applied the method to study the effect of SHP2, a protein that has been implicated in resistance to targeted therapy in colon cancer, using a novel dataset from the colon cancer cell line Widr and a SHP2-depleted derivative. We find that SHP2 is required for mitogen-activated protein kinase signalling, whereas AKT signalling only partially depends on SHP2. An R-package is available at https://github.com/molsysbio/STASNet. Supplementary data are available at Bioinformatics online.

Pentagamavunone-0 (PGV-0), a Curcumin Analog, Enhances Cytotoxicity of 5-Fluorouracil and Modulates Cell Cycle in WiDr Colon Cancer Cells

The use of 5-fluorouracil (5-FU) in colon cancer as the primary chemotherapy has not been meet satisfactory effectiveness. Therefore, the development of new chemicals as a chemopreventive agent and a combination agent (co-chemotherapeutic agent) for colon cancer is important. Pentagamavunone-0 (2,5-bis-(4'-hydroxy-3'-methoxybenzylidine) cyclopentanone) (PGV-0), one of curcumin analogs, exhibits cytotoxic effect and apoptosis induction in various cancer cell lines, including colon cancer cell, better than curcumin. This study aimed to investigate the cytotoxic potency of PGV-0 in combination with 5-FU and their effects, in single or in combination, on cell cycle toward WiDr colon cancer cell line. The cells were treated with combination concentrations of PGV-0 and 5-FU, and examined by MTT cell viability assay. The value of combination index (CI) as a parameter of cytotoxic combination assay was measured by a combination index method. Cells were stained with propidium iodide and the cell cycle distribution was determined by flowcytometry. CI calculation showed additive effects between PGV-0 and 5-FU. Combination of PGV-0 and 5-FU gave synergism on cell cycle. Single treatment of PGV-0 increased apoptosis, illustrated as subG1-phase accumulation, stronger than single treatment of 5-FU. Meanwhile, combination of PGV-0 and 5-FU demonstrated S-phase arrest. Based on these results, it can be concluded that PGV-0 has the potential to be developed as a co-chemotherapeutic agent for colon cancer but still requires further tracking of its molecular mechanisms.Keywords: Pentagamavunone-0 (PGV-0), 5-fluorouracil (5-FU), colon cancer,combination, cell cycle

Interleukin-1 receptor antagonist inhibits angiogenesis via blockage IL-1α/PI3K/NF-κβ pathway in human colon cancer cell.

PurposeThis article investigates the relationship between cancer cells and stromal cells in carcinoma cell living microenvironment and elucidates the mechanism that interleukin-1 receptor antagonist (IL-1RA) blocks metastatic potential in colon cancer.MethodsWestern blot and RT-PCR assay were used to determine the expression of hepatocyte growth factor (HGF) and IL-1α in colon carcinoma cells and stromal cells. Effect of IL-1RA and HGF on metastatic potential of colon cancer cells were examined by proliferation, invasion, and angiogenesis assays. The interactional role of IL-1RA and HGF were detected by ELISA assay, invasion, and angiogenesis assay making up a co-culture system consisting of stromal and colon cancer cells in cells living microenvironment.ResultsIL-1α was expressed in human umbilical vein endothelial cells (HUVECs) and HT-29 and WiDr (colon cancer cell lines with higher liver metastatic potential). HGF was expressed only in fibroblast. HGF secretion from fibroblasts was significantly inhibited by IL-1RA (P<0.01). Furthermore, IL-1RA could significantly inhibit migration, proliferation, and angiogenesis of HUVECs (P<0.01). In the double co-culture system, there is a high liver metastatic potential of colon cancer cell line (HT-29) because it can secrete autocrine IL-1α, significantly enhanced angiogenesis compared with low liver metastatic cell line (CaCo-2) (P<0.01), which does not secrete IL-1α. On the contrary, blockage of autocrine IL-1α by IL-1RA might significantly decrease metastatic potential of colon carcinoma cells through downregulation of IL-1α/PI3K/NF-κB pathway.ConclusionIL-1 receptor antagonist (IL-1RA) is an important inhibitor in metastatic process of colon carcinoma cell. Based on the above results, we suggest that IL-1RA may be a promising new therapeutic approach in inhibiting colon cancer with IL-1-producing patients.

Synthesis and in vitro antiproliferative activities of (5-aryl-1,2,4-oxadiazole-3-yl) methyl d-ribofuranosides

The emergence of multidrug resistance cell lines is one of the major obstacles in the success of cancer chemotherapeutic treatment. Therefore, it remains a big challenge the development of new and effective drugs to defeat cancer. The presence of nitrogen heterocycles in the architectural design of drugs has led to the discovery of new leading compounds. Herein, we report the synthesis, characterization and in vitro antiproliferative activity against six cancer cell lines of d-ribofuranoside derivatives bearing a 1,2,4-oxadiazolic ring, with the aim of developing new active compounds. Most of these derivatives exhibit significant antiproliferative activities in the micromolar range. Noteworthy, the most potent compound of the series showed better selectivity towards the more resistant colon cancer cell line WiDr.

Antiproliferative Activity and Effect on GABAA Receptors of Callitrisic Acid Derivatives

AbstractThe semisynthesis and biological activity of the naturally occurring abietane diterpenoids callitrisic acid (4a; 4-epidehydroabietic acid) and callitrisinol (6) are reported. These compounds and jiadifenoic acid C (5) were obtained from methyl callitrisate (4b) isolated from Sandarac resin for biological evaluation and comparison with the biological activities of C4 epimers such as dehydroabietic acid (2a). In particular, the antiproliferative activity against a panel of six representative human solid tumor cell lines (A549, HBL-100, HeLa, SW1573, T-47D, WiDr) and the effect on GABAA receptors (α 1 β 2 γ 2s) were evaluated. The GI50 values were in the range of 3.4–61 µM and the potentiation of IGABA was 269–311% at 100 µM. Callitrisinol (6) was found to be 6.7-fold more potent than the reference etoposide in the WiDr (colon) cancer cell line. The role of the stereogenic center at C4 for antiproliferative and GABAA receptor modulating activities in the dehydroabietane scaffold has thus been revealed.

Cytotoxic Activity of 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone on Colon Cancer Cell WiDr

Colon cancer is one of the most common death-caused cancer. The high mortality rate indicates that chemotherapy has not overcome cancer disease. Strategies and development of colon cancer treatment should be pursued. Compound 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone is the 2ʹ,5ʹ-dihydroxychalcone derivative, of which the B ring was substituted with 2-pyridine ring. Chalcone and its derivatives have been reported to have several biological activities, such as cytotoxic, anti-inflammatory, antiHIV, and as a tyrosine kinase inhibitor. The objectives of this research was to determine the cytotoxic activity on WiDr colon cancer cells of 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone. Cytotoxic activity was measured using MTT assay. Compound 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone inhibited WiDr cell growth with the IC50 of 16 µM. Morphology of WiDr cell showed that compound 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone inhibited cell growth in dose dependent.Keywords: compound 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone, WiDr colon cancer cell line, cytotoxic activity

The expression of multidrug resistance protein 5 and thymydilate synthase on fluorouracil resistance WiDr colon cancer cell line

Background : Colorectal cancer becomes one of the most common cancer in Indonesia. Fluorouracil is an essential drug in colorectal cancer therapy as it has been supplemented in about 69% of current regiments of chemotherapy. However, drug resistance has reduced its clinical applications. Many factors that contribute to fluorouracil resistance have been investigated including overexpression of Multidrug Resistance Protein 5 (MRP 5) and high level expression of Thymidylate Synthase (TS). Therefore, the expression of those proteins may become key indicators to predict the presence of resistance in the model of fluorouracil-resistance-WiDr colon cancer cell line. Objective: The aim of this study is to characterize a 5-FU acquired resistance WiDr colon cancer cell line, focus on the expression of MRP5 and TS. Methods: This study was a post test controlled group design. The expression of MRP5 and TS were analyzed using immunocytochemistry method. Before protein expression analysis, 4 groups of WiDr colon cancer cell line, group 1, 2, 3, and 4 (control) were treated with 4 different concentration of fluorouracil 12.3 I¼M, 6.15 I¼M, 3.08 I¼M, and 0 I¼M respectively. Previous study showed that a significant increased level of IC50 value is observed in group induced with fluorouracil 3.08 I¼M (group 3). Statistical tests for protein expression performed with SPSS version 19 using Kruskall Wallis with 95% confidence level (p<0.05). Results: The expression of MRP5 and TS in the group developed fluorouracil resistance (group 3) were significantly increased compared to control group (p<0.05). In contrast, the other group exposed with higher dose of fluorouracil did not show both significant increased level of IC50 nor significant different of MRP5 and TS expression compared with the control group. Conclusion: The model of fluorouracil-acquired resistant WiDr cell line expresses Thymidilate Synthase and Multidrug Resistance Protein 5. The expressions of these proteins are in accordance with the profile of fluorouracil resistancy.

P0100 Upregulation of Bax in colon cancer cell line WiDr p53−/− when treated with metformin in different concentrations in vitro

Background Metformin has been associated with antitumour activity in epidemiological and clinical studies. This effect has been related to different mechanisms of actions including an increase in apoptosis. To investigate the effects of different metformin concentrations on expression of pro-apoptotic protein Bax in the colon cancer cell line WiDr. Methods Colon cancer cell line WiDr p53−/− was obtained from Parasitology Laboratory of Gadjah Mada University. Cells were cultured in RPMI supplemented and 1% penicillin at 5% CO2, divided into four groups. Each group was treated with 0 (control), 5, 10, or 20 mmol/l metformin and incubated for 48 h at 37°C. Viable cell ratio in each study group was obtained by MTT test at 540 nm microelisa reader. Bax immunostaining was applied to detect cell apoptosis and was scored using the semiquantitative H-score, which takes into consideration the staining intensity (0–3 or more) in conjunction with the percentage of viable tumour cells that stained positive. Viable cell ratios were evaluated by ANOVA test. Data were presented as arithmetic mean ± SD. H-score of Bax immunostaining was assessed using non-parametrical tests. p-values less than 0.05 were statistically significant. Findings Viable cell count in the study groups (70.20 ± 11.08 [5 mmol/l], 67.93 ± 12.95 [10 mmol/l], 58.37 ± 13.08 [20 mmol/l]) were low compared with the control group. Bax was significantly upregulated in 5 mmol/l (p = 0.019), 10 mmol/l (p = 0.031), and 20 mmol/l (p = 0.002) metformin treated cells compared with the control group. Interpretation Metformin could increase apoptotic activity by upregulating Bax independent of p53. Metformin has been associated with antitumour activity in epidemiological and clinical studies. This effect has been related to different mechanisms of actions including an increase in apoptosis. To investigate the effects of different metformin concentrations on expression of pro-apoptotic protein Bax in the colon cancer cell line WiDr. Colon cancer cell line WiDr p53−/− was obtained from Parasitology Laboratory of Gadjah Mada University. Cells were cultured in RPMI supplemented and 1% penicillin at 5% CO2, divided into four groups. Each group was treated with 0 (control), 5, 10, or 20 mmol/l metformin and incubated for 48 h at 37°C. Viable cell ratio in each study group was obtained by MTT test at 540 nm microelisa reader. Bax immunostaining was applied to detect cell apoptosis and was scored using the semiquantitative H-score, which takes into consideration the staining intensity (0–3 or more) in conjunction with the percentage of viable tumour cells that stained positive. Viable cell ratios were evaluated by ANOVA test. Data were presented as arithmetic mean ± SD. H-score of Bax immunostaining was assessed using non-parametrical tests. p-values less than 0.05 were statistically significant. Viable cell count in the study groups (70.20 ± 11.08 [5 mmol/l], 67.93 ± 12.95 [10 mmol/l], 58.37 ± 13.08 [20 mmol/l]) were low compared with the control group. Bax was significantly upregulated in 5 mmol/l (p = 0.019), 10 mmol/l (p = 0.031), and 20 mmol/l (p = 0.002) metformin treated cells compared with the control group. Metformin could increase apoptotic activity by upregulating Bax independent of p53.

P0101 Metformin inhibits cyclin D1 expression in a p53-deficient colon cancer cell line in vitro

Background Metformin is a biguanide that is used as first-line therapy for diabetes mellitus. It regulates metabolic profiles in the treatment of diabetes mellitus. Metformin has a potential anticancer effect, which could be helpful for patients with metabolic-related cancers such as colon cancer. This study aims to explore the anticancer role of metformin in the inhibition of cyclin D1 as a regulator of the cell cycle and to analyse the molecular target of metformin in colon cancer. Methods WiDr colon cancer cell lines were obtained from the parasitology laboratory of Gadjah Mada University. WiDr cell lines were treated with metformin at concentrations of 5 mmol/l, 10 mmol/l, and 20 mmol/l and incubated in 37 °C and 5% CO2 conditions. MTT assays were done to assess tumour-cell viability. Subsequently, we used immunohistochemistry to measure cyclin D1 expression. Cyclin D1 expression was quantified by H score using percentage and intensity for calculation. ANOVA was used to compare cell viability between groups. Comparisons of H score between groups were performed by Kruskal–Wallis for non-parametric tests. Findings Metformin significantly decreased cell viability at all concentrations by 70.20 ± 11.08 (p = 0.009), 67.93 ± 12.95 (p = 0.006), and 58.37 ± 13.08 (p = 0.001), respectively. Subsequently, metformin significantly down-regulated cyclin D1 expression at 10 mmol/l (p = 0.031) and 20 mmol/l (p = 0.0001) concentrations. Interpretation Our results suggest that metformin could inhibit colon cancer proliferation in p53 independent cell lines. Cyclin D1 might be a molecular target of metformin in p53-mutant colon cancer. Metformin is a biguanide that is used as first-line therapy for diabetes mellitus. It regulates metabolic profiles in the treatment of diabetes mellitus. Metformin has a potential anticancer effect, which could be helpful for patients with metabolic-related cancers such as colon cancer. This study aims to explore the anticancer role of metformin in the inhibition of cyclin D1 as a regulator of the cell cycle and to analyse the molecular target of metformin in colon cancer. WiDr colon cancer cell lines were obtained from the parasitology laboratory of Gadjah Mada University. WiDr cell lines were treated with metformin at concentrations of 5 mmol/l, 10 mmol/l, and 20 mmol/l and incubated in 37 °C and 5% CO2 conditions. MTT assays were done to assess tumour-cell viability. Subsequently, we used immunohistochemistry to measure cyclin D1 expression. Cyclin D1 expression was quantified by H score using percentage and intensity for calculation. ANOVA was used to compare cell viability between groups. Comparisons of H score between groups were performed by Kruskal–Wallis for non-parametric tests. Metformin significantly decreased cell viability at all concentrations by 70.20 ± 11.08 (p = 0.009), 67.93 ± 12.95 (p = 0.006), and 58.37 ± 13.08 (p = 0.001), respectively. Subsequently, metformin significantly down-regulated cyclin D1 expression at 10 mmol/l (p = 0.031) and 20 mmol/l (p = 0.0001) concentrations. Our results suggest that metformin could inhibit colon cancer proliferation in p53 independent cell lines. Cyclin D1 might be a molecular target of metformin in p53-mutant colon cancer.

The effectiveness of an anti-human IL-6 receptor monoclonal antibody combined with chemotherapy to target colon cancer stem-like cells.

Recent studies have demonstrated that cancer stem cells (CSCs) can initiate and sustain tumor growth and exhibit resistance to clinical cytotoxic therapies. Therefore, CSCs represent the main target of anticancer therapy. Interleukin-6 (IL-6) promotes cellular proliferation and drug resistance in colorectal cancer, and its serum levels correlate with patient survival. Therefore, IL-6 and its downstream signaling molecule the signal transducer and activator of transcription-3 (STAT3) represent potential molecular targets. In the present study, we investigated the effects of IL-6 and its downstream signaling components on stem cell biology, particularly the chemoresistance of CSCs, to explore potential molecular targets for cancer therapy. The colon cancer cell line WiDr was cultured in serum-free, non-adherent, and three-dimensional spheroid-forming conditions to enrich the stem cell-like population. Spheroid-forming cells slowly proliferated and expressed high levels of Oct-4, Klf4, Bmi-1, Lgr5, IL-6, and Notch 3 compared with adherent cells. Treatment with an anti-human IL-6 receptor monoclonal antibody reduced spheroid formation, stem cell-related gene expression, and 5-fluorouracil (5-FU) resistance. In addition, IL-6 treatment enhanced the levels of p-STAT3 (Tyr705), the expression of Oct-4, Klf4, Lgr5, and Notch 3, and chemoresistance to 5-FU. siRNA targeting Notch 3 suppressed spheroid formation, Oct-4 and Lgr5 expression, and 5-FU chemoresistance, whereas STAT3 inhibition enhanced Oct-4, Klf4, Lgr5, and Notch 3 expression and 5-FU chemoresistance along with reduced spheroid growth. Taken together, these results indicate that IL-6 functions in dichotomous pathways involving Notch 3 induction and STAT3 activation. The former pathway is involved in cancer stem-like cell biology and enhanced chemoresistance, and the latter pathway leads to accelerated proliferation and reduced chemoresistance. Thus, an anti-human IL-6 receptor monoclonal antibody or Notch 3 inhibition may be superior to STAT3 inhibition for CSC-targeting therapies concomitant with anticancer drugs.