Colorectal Cancer Cell Lines HCT116 Research Articles

Fatty acid composition, enzyme inhibitory effect, antioxidant and anticancer activity of extract from Saponaria prostrata WILLD. subsp. anatolica HEDGE

This study attempts to evaluate the antioxidant, enzyme inhibitory, and anticancer properties as well as fatty acid compositions of endemic Saponaria prostrata WILLD. subsp. anatolica HEDGE. The gas chromatography-mass spectrometry (GC–MS) was used to determine the fatty acid content of methanol: dichloromethane extract from S. prostrata subsp. anatolica (SPA). Enzymatic activity was measured against acetylcholinesterase, butyrylcholinesterase and α-glucosidase. DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity and Ferric reducing antioxidant power assay (FRAP) were conducted to antioxidant properties. The anticancer effect of SPA on human MCF-7 breast cancer and human HCT116 colorectal cancer cell line was evaluated by WST-1 cell viability assay, colony formation assay and wound healing assay. In addition, human VEGF Elisa method was used to determine the anti-angiogenic effect, and the quantitative real-time PCR (qRT-PCR) method on p53, Bax and Bcl-2 mRNA levels were used to evaluate apoptosis. While high amounts of palmitic acid (40.8%), linoleic acid (17.75%) and α-linolenic acid (16.84%) were detected in the SPA, the total amount of unsaturated fatty acid (51.34%) was higher than the total amount of saturated fatty acid (48.66%). SPA displayed the most promising acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and α-glycosidase (AG) inhibitory activities (AChE: IC50: 18.03 µg/mL, BuChE: IC50: 44.24 µg/mL and AG: IC50: 210.85 µg/mL). The half maximum inhibitory concentration (IC50) of SPA in MCF-7 and HCT116 cells was determined as 259.79 µg/mL and 97.24 µg/mL, respectively. In addition, it was determined that SPA suppresses colony formation and wound closure, and suppresses angiogenesis as well as triggering apoptosis at a significant level. It is true that endemic S. prostrata subsp. anatolica is a potential source of functional food ingredients, but more analytical and in vivo experiments are needed to explore further secondary metabolite diversity and pharmacological properties.

New tropolone derivative to inhibit cell motility in colorectal cancer cells.

e15043 Background: Tropolones are small, naturally occurring organic compounds that have anti-cancer effects in a variety of cancer cell lines. Among proposed mechanisms of tropolone derivatives anti-cancer activity are caspase activation, matrix metalloproteinases and histone deacetylases inhibition and other actions. Since matrix metalloproteinases are the major proteins that contribute to tumor cell tissue invasion and migration their blockers are extensively studied as prospective anti metastatic agents. We assumed that tropolones may also have potential as metastases prevention drugs. The aim of the study was to investigate the influence of new tropolone derivative 2-(6,8-dimethyl-5-nitro-4-chloroquinolin-2-yl)-5,6,7-trichloro-1,3-tropolone on the cell migration capacity in colorectal cancer cells. Methods: Cell migration in permanent colorectal cancer cell lines HT29, Caco2 and HCT116 was studied using the standard wound healing assay with automated image acquisition and analysis on Lionheart FX (BioTek) cell imager. Cell cultures were maintained in DMEM medium with L-glutamine (1 μM) (Gibco) and 10% FBS (Gibco) at 37C0 and 5.0% CO2. The day before assay establishment cells were seeded at 250 000 cells per well on 24-well plates. After attachment of cells, a vertical wound in cell layer was made manually in each well, and then medium with studied tropolone derivative (5 μM) was added. Plates with cells were continuously incubated and photographed in cell imager at 37C0 and 5.0% CO2. The extent of cells migration was measured as the percent of wound area decrease after 48 hours of incubation in relation to starting time point. Data are given as: Mean ± 95% confidence interval. Results: The new tropolone derivative under study exhibited pronounced anti migratory activity in all studied colorectal cancer cell cultures. The most prominent effect was observed in HCT116 cell line: after 48 hours of incubation relative wound area decrease was 48.2%±5.7% in control and 7.2%±3.15% in experimental groups. In Caco2 cell line cell migration inhibition was the least with 65%± 6.9% relative wound area decrease in control and 51.5%± 7.7% in experimental groups. The moderate effect was observed in HT29 cell line (measured parameter was 63.6%±7.6% and 36.9%±11.6% in control and experimental groups accordingly). The obtained difference between control and experimental groups was significant in all tested cell cultures at 0.05 level (df = 30). Conclusions: Results of the present study prove tropolone derivative to be prospective candidates for anti-metastatic drug development. Further investigations are needed to reveal the exact mechanisms of tropolones anti-migratory activity.

MiRNA-mRNA Regulatory Network Reveals miRNAs in HCT116 in Response to Folic Acid Deficiency via Regulating Vital Genes of Endoplasmic Reticulum Stress Pathway.

Moderate folic acid (FA) intake is an effective strategy that slows colorectal cancer (CRC) progression. However, high consumption of FA may trigger the transition of precancerous tissue towards malignancy. MicroRNAs (miRNAs) are considered to be potential biomarkers of CRC. Thus, identification of miRNAs of dysregulated genes in CRC cells by detailed analysis of mRNA and miRNA expression profile in the context of FA deficiency could substantially increase our understanding of its oncogenesis. mRNA-seq and miRNA-seq analyses were utilized to investigate the expression of miRNAs in FA-deficient CRC cell line–HCT116 through massive parallel sequencing technology. A total of 38 mRNAs and 168 miRNAs were identified to be differentially expressed between CRC groups with or without FA deficiency. We constructed an miRNA-mRNA network for the vital regulatory miRNAs altered in FA-deficient CRC cells. The mRNAs and miRNAs validated by Western blotting and RT-qPCR were consistent with the sequencing results. Results showed that FA deficiency upregulated some miRNAs thereby inhibiting the expression of critical genes in the endoplasmic reticulum (ER) stress pathway. Dysregulated miRNAs in our miRNA-mRNA network could contribute to CRC cell in response to deficient FA. This work reveals novel molecular targets that are likely to provide therapeutic interventions for CRC.

Synthesis of diosgenin conjugated gold nanoparticles using algal extract of Dictyosphaerium sp. and in-vitro application of their antiproliferative activities

In this study, gold nanoparticles (AuNPs) and novel gold nanoformulations loaded with diosgenin (Dio-AuNPs) were synthesized through green approach using algal extract from Dictyosphaerium sp. strain DHM2 (LC159306) as a reducing agent. AuNPs and Dio-AuNPs synthesized were characterized through UV–vis spectroscopy, zeta potential analysis, FT-IR spectroscopy, X-ray diffraction (XRD) analysis and scanning transmission electron miroscopy. An average zeta potential value of -32.4 mV indicated that AuNPs were highly stable. Fourier transmission infrared spectroscopy (FT-IR) of AuNPs showed the presence of hydroxyl groups and carbonyl groups alongwith various other capping and stabilizing functional groups. Also, in FT-IR spectrum of Dio-AuNPs characteristic peaks of both AuNPs and diosgenin were observed. XRD analysis of AuNPs and Dio-AuNPs showed that both nanostuctures exist in the crystalline form. Scanning transmission electron microscopy (STEM) studies revealed that AuNPs and Dio-AuNPs had spherical morphology with average size range of 7.9 ± 2.4 nm and 10.3 ± 2.4 nm, respectively. Moreover, in-vitro antiproliferative activities of AuNPs and Dio-AuNPs were determined on colorectal cancer cell line HCT116 and breast cancer cell line HCC1954 using sulforhodamine B (SRB) proliferation assay. Dio-AuNPs were extremely potent with GI50 values of 1.03 ± 0.27 μg/mL and 1.69 ± 0.18 μg/mL in HCT116 and HCC1954 cell lines, respectively. AuNPs however, were almost 18 fold less potent as compared to Dio-AuNPs in both the cell lines. The current study has demonstrated biological synthesis, characterization and antiproliferative activities of gold nanoparticles and novel diosgenin loaded gold nanoformulations in colorectal and breast cancer cell lines and provided evidence that anticancer activities of both diosgenin and AuNPs can be enhanced many folds when used in conjugation.

Abstract PO-026: DNA damage response and repair characterization in CHK2-deficient cancers

Abstract Double strand breaks (DSBs) are the most lethal DNA lesions as they can result in chromosomal rearrangements or loss of genetic material. To detect and repair DNA, cells have developed the DNA damage response, a cascade of proteins in which the checkpoint kinase 2 (CHK2) is involved. CHK2-deficient cancers, such as breast, prostate or colon, might have a compromised response to ionizing radiation (IR), arresting cell cycle at checkpoints or inducing apoptosis in presence of irreparable damage. To understand how the lack of CHK2 is affecting DNA damage response and repair, the human isogenic HCT116 colorectal cancer cell lines proficient (CHK2 WT) and deficient (CHK2 KO) for CHK2 have been characterized. Cell cycle distribution and γH2AX foci formation were assessed by flow cytometry and immunofluorescence, respectively, to determine the differences in cell cycle checkpoints and DSBs infliction and resolution upon IR. Traffic Light Reporter, complemented by immunofluorescence of RAD51 foci, was employed in both CHK2 WT and KO cell lines to identify the preferred DSB repair pathway. Our results indicate that CHK2-proficient and -deficient cell lines do not exhibit significant differences in cell cycle distribution upon IR. On the other hand, CHK2 KO cells display a significant increase in γH2AX foci per cell compared to the CHK2 WT. Upon 1Gy of IR, cells lacking CHK2 increment the number of foci per cell, indicating that DSBs detection occurs independently of the CHK2 status. Furthermore, the Traffic Light Reporter revealed higher homologous recombination (HR) to non-homologous end-joining (NHEJ) ratio in the CHK2 KO cells as compared to their CHK2 WT counterparts, suggesting that the lack of CHK2 stimulates a shift in the DSBs repair pathway choice. These results contrast with the RAD51 foci formation, which seems to be compromised in the CHK2 KO cells in both treated and untreated conditions as compared to the CHK2-proficient cells. Our results suggest that CHK2 status considerably affects cellular DNA damage response and these differences in execution of DNA repair could be exploited in CHK2-deficient cancers. Citation Format: Carmen Muñoz-Maldonado, Aurélie Quintin, Daniel M. Aebersold, Yitzhak Zimmer, Michaela Medová. DNA damage response and repair characterization in CHK2-deficient cancers [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-026.

Metformin Inhibits the Urea Cycle and Reduces Putrescine Generation in Colorectal Cancer Cell Lines.

The urea cycle (UC) removes the excess nitrogen and ammonia generated by nitrogen-containing compound composites or protein breakdown in the human body. Research has shown that changes in UC enzymes are not only related to tumorigenesis and tumor development but also associated with poor survival in hepatocellular, breast, and colorectal cancers (CRC), etc. Cytoplasmic ornithine, the intermediate product of the urea cycle, is a specific substrate for ornithine decarboxylase (ODC, also known as ODC1) for the production of putrescine and is required for tumor growth. Polyamines (spermidine, spermine, and their precursor putrescine) play central roles in more than half of the steps of colorectal tumorigenesis. Given the close connection between polyamines and cancer, the regulation of polyamine metabolic pathways has attracted attention regarding the mechanisms of action of chemical drugs used to prevent CRC, as the drug most widely used for treating type 2 diabetes (T2D), metformin (Met) exhibits antitumor activity against a variety of cancer cells, with a vaguely defined mechanism. In addition, the influence of metformin on the UC and putrescine generation in colorectal cancer has remained unclear. In our study, we investigated the effect of metformin on the UC and putrescine generation of CRC in vivo and in vitro and elucidated the underlying mechanisms. In nude mice bearing HCT116 tumor xenografts, the administration of metformin inhibited tumor growth without affecting body weight. In addition, metformin treatment increased the expression of monophosphate (AMP)-activated protein kinase (AMPK) and p53 in both HCT116 xenografts and colorectal cancer cell lines and decreased the expression of the urea cycle enzymes, including carbamoyl phosphate synthase 1 (CPS1), arginase 1 (ARG1), ornithine trans-carbamylase (OTC), and ODC. The putrescine levels in both HCT116 xenografts and HCT116 cells decreased after metformin treatment. These results demonstrate that metformin inhibited CRC cell proliferation via activating AMPK/p53 and that there was an association between metformin, urea cycle inhibition and a reduction in putrescine generation.

Structurally diverse triterpenoids with cytotoxicity from Euphorbia hypericifolia

Extensive phytochemical investigation on the whole herbs of Euphorbia hypericifolia led to the isolation of 18 structurally diverse tetracyclic and pentacyclic triterpenoids, including four 4α,14α-dimethyl-5α-ergostanes (1–4), two seco-adiananes (5 and 6), three dammaranes (7–9), four cycloartanes (10–13), one tirucallane (14), two fernanes (15 and 16), one ursane (17), and one oleanane (18). Among them, euphypenoids A (1) and B (5) were new triterpenoids. Their structures were elucidated on the basis of extensive spectroscopic analysis, single-crystal X-ray diffraction, and chemical transformation. All isolates were screened for their cytotoxic activities against the colorectal cancer cell line HCT-116, and compounds 1, 12, and 15 showed remarkable activities with IC50 values of 12.8 ± 1.6, 7.4 ± 0.2, and 10.6 ± 1.2 μM, respectively.

Anti-migration Effect of Aaptos suberitoides Fraction in HCT-116 Colorectal Cancer Cell Line

Background: Colorectal cancer is the second leading cause of mortality and the most prevalent cancer worldwide. Most patients, who come with late-stage, have ineffective treatments and some side effects in chemotherapy. Aaptos suberitoides has potential anti-cancer effects due to its bioactive compounds such as aptamine. This study aimed to evaluate the migration inhibition effect of Aaptos suberitoides fraction in HCT-116 cell line.Methods: This study was an experimental study. Aaptos suberitoides specimen was taken in Tinjil Island and fractionated with ethyl acetate. HCT-116 cell line was added with Aaptos suberitoides fraction and cellular migration activity was observed in 48 hours of which the scratch assay was performed. The gap closure area was determined with ImageJ software.Results: The data showed that a low concentration of Aaptos suberitoides fraction inhibited migration activity in HCT-116 cell line as follow; 1 and 5 mg/L Aaptos suberitoides fraction inhibit 3-4 % cancer cell migration in 24 hours, and 10-11% inhibition in 48 hours, respectively. However, 10 mg/L fraction concentration only inhibited 7-14% of the migration effect.Conclusion: Aaptos suberitoides fraction suggests insignificant migration inhibition in colorectal cancer cells and only inhibits less than 15 % HCT-116 cell line.

In oxygen-deprived tumor cells ERp57 provides radioprotection and ensures proliferation via c-Myc, PLK1 and the AKT pathway

The disulfide isomerase ERp57, originally found in the endoplasmic reticulum, is located in multiple cellular compartments, participates in diverse cell functions and interacts with a huge network of binding partners. It was recently suggested as an attractive new target for cancer therapy due to its critical role in tumor cell proliferation. Since a major bottleneck in cancer treatment is the occurrence of hypoxic areas in solid tumors, the role of ERp57 in cell growth was tested under oxygen depletion in the colorectal cancer cell line HCT116. We observed a severe growth inhibition when ERp57 was knocked down in hypoxia (1% O2) as a consequence of downregulated c-Myc, PLK1, PDPK1 (PDK1) and AKT (PKB). Further, irradiation experiments revealed also a radiosensitizing effect of ERp57 depletion under oxygen deprivation. Compared to ERp57, we do not favour PDPK1 as a suitable pharmaceutical target as its efficient knockdown/chemical inhibition did not show an inhibitory effect on proliferation.

Autophagy inhibitors increase the susceptibility of KRAS-mutant human colorectal cancer cells to a combined treatment of 2-deoxy-D-glucose and lovastatin.

RAS-driven colorectal cancer relies on glucose metabolism to support uncontrolled growth. However, monotherapy with glycolysis inhibitors like 2-deoxy-D-glucose causes limited effectiveness. Recent studies suggest that anti-tumor effects of glycolysis inhibition could be improved by combination treatment with inhibitors of oxidative phosphorylation. In this study we investigated the effect of a combination of 2-deoxy-D-glucose with lovastatin (a known inhibitor of mevalonate pathway and oxidative phosphorylation) on growth of KRAS-mutant human colorectal cancer cell lines HCT116 and LoVo. A combination of lovastatin (>3.75 μM) and 2-deoxy-D-glucose (>1.25 mM) synergistically reduced cell viability, arrested cells in the G2/M phase, and induced apoptosis. The combined treatment also reduced cellular oxygen consumption and extracellular acidification rate, resulting in decreased production of ATP and lower steady-state ATP levels. Energy depletion markedly activated AMPK, inhibited mTOR and RAS signaling pathways, eventually inducing autophagy, the cellular pro-survival process under metabolic stress, whereas inhibition of autophagy by chloroquine (6.25 μM) enhanced the cytotoxic effect of the combination of lovastatin and 2-deoxy-D-glucose. These in vitro experiment results were reproduced in a nude mouse xenograft model of HCT116 cells. Our findings suggest that concurrently targeting glycolysis, oxidative phosphorylation, and autophagy may be a promising regimen for the management of RAS-driven colorectal cancers.

Activation of WNT7b autocrine eases metastasis of colorectal cancer via epithelial to mesenchymal transition and predicts poor prognosis

BackgroundAberrant activation of the Wnt/β-catenin signaling pathway is one of the most frequent abnormalities in human cancer, including colorectal cancer (CRC). Previous studies revealed pivotal functions of WNT family members in colorectal cancer, as well as their prognostic values. Nevertheless, the prognostic role and mechanisms underlying WNT7b in colorectal cancer development remains unclear.MethodsIn this study, WNT7b expression was measured by immunohistochemical staining of 100 cases of surgically resected human colorectal cancerous tissues as well as matched adjacent normal tissues constructed as tissue microarrays. In vitro studies, we attempted to substantiate the WNT7b expressional pattern previously found in immunohistochemistry staining. We used the colorectal cancer cell-line HCT116 and normal colorectal cell-line FHC for immunofluorescence staining and nuclear/cytoplasmic separated western blotting. We measured epithelial–mesenchymal transition (EMT) markers and migration capacity of HCT116 in the context of WNT7b knocked-down using short interfering RNA. Finally, clinical and prognostic values of WNT7b activation levels were examined.ResultsWNT7b was expressed in the nucleus in adjacent normal tissues. In CRC tissues, nuclear expression of WNT7b was similar; however, membrane and cytoplasmic expression was strikingly enhanced. Consistently, in vitro analysis confirmed the same expression pattern of WNT7b. Compared with FHC cells, HCT116 cells displayed higher levels of WNT7b membrane and cytoplasmic enrichment, as well as higher migration capacity with a sensitized EMT process. Either partial knockdown of WNT7b or blockade of the Wnt/β-catenin signaling pathway reversed EMT process and inhibited the migration of HCT116 cells. Finally, elevated secretion levels of WNT7b were significantly associated with lymphatic and remote metastasis and predicted worse prognosis in the CRC cohort.ConclusionIn summary, we demonstrated that the activation of WNT7b autocrine probably contributes to CRC metastasis by triggering EMT process through the Wnt/β-catenin signaling pathway. High levels of WNT7b autocrine secretion predicts poor outcome in patients with CRC. This molecule is a promising candidate for clinical CRC treatments.

Fangchinoline exerts anticancer effects on colorectal cancer by inducing autophagy via regulation AMPK/mTOR/ULK1 pathway

Autophagy has become a promising target for cancer therapy. Fangchinoline (Fan) has been shown to exert anticancer effects in some types of cancers. However, the anticancer effects on colorectal cancer (CRC) and the underlying mechanisms have never been elucidated. More specifically, regulation of autophagy in CRC by Fan has never been reported before. In the present study, Fan was found to induce apoptosis and autophagic flux in the CRC cell lines HT29 and HCT116, which was reflected by the enhanced levels of LC3-II protein and p62 degradation, and the increased formation of autophagosomes and puncta formation by LC3-II. Meanwhile, combination with the early-stage autophagy inhibitor 3-methyladenine (3-MA) but not the late-stage autophagy inhibitor chloroquine (CQ) further increased Fan-induced cell death, which suggested the cytoprotective function of autophagy induced by Fan in both HT29 and HCT116 cells. Moreover, Fan treatment demonstrated a dose- and time-dependently increase in the phosphorylation of AMPK and decrease in the phosphorylation of mammalian target of rapamycin (mTOR) and ULK1, leading to the activation of the AMPK/mTOR/ULK1 signaling pathway. Furthermore, in the HT29 xenograft model, Fan inhibited tumor growth in vivo. These results indicate that Fan inhibited CRC cell growth both in vitro and in vivo and revealed a new molecular mechanism involved in the anticancer effect of Fan on CRC, suggesting that Fan is a potent autophagy inducer and might be a promising anticancer agent.

Biguanide-Based Synthesis of 1,3,5-Triazine Derivatives with Anticancer Activity and 1,3,5-Triazine Incorporated Calcium Citrate Nanoparticles.

Twelve derivatives of biguanide-derived 1,3,5-triazines, a promising class of anticancer agent, were synthesised and evaluated for their anticancer activity against two colorectal cancer cell lines—HCT116 and SW620. 2c and 3c which are the derivatives containing o-hydroxyphenyl substituents exhibited the highest activity with IC50 against both cell lines in the range of 20–27 µM, which is comparable to the IC50 of cisplatin reference. Moreover, the potential use of the calcium citrate nanoparticles (CaCit NPs) as a platform for drug delivery system was studied on a selected 1,3,5-triazine derivative 2a. Condition optimisation revealed that the source of citrate ions and reaction time significantly influence the morphology, size and %drug loading of the particles. With the optimised conditions, “CaCit-2a NPs” were successfully synthesised with the size of 148 ± 23 nm and %drug loading of up to 16.3%. Furthermore, it was found that the release of 2a from the synthesised CaCit-2a NPs is pH-responsive, and 2a could be control released under the acidic cancer environment. The knowledge from this study is perceptive for further development of the 1,3,5-triazine-based anticancer drugs and provide the platform for the incorporation of other drugs in the CaCit NPs in the future.

Negative Regulation of ULK1 by microRNA-106a in Autophagy Induced by a Triple Drug Combination in Colorectal Cancer Cells In Vitro

Colorectal cancer (CRC) is among the top three most deadly cancers worldwide. The survival rate for this disease has not been reduced despite the treatments, the reason why the search for therapeutic alternatives continues to be a priority issue in oncology. In this research work, we tested our successful pharmacological combination of three drugs, metformin, doxorubicin, and sodium oxamate (triple therapy, or TT), as an autophagy inducer. Firstly, we employed western blot (WB) assays, where we observed that after 8 h of stimulation with TT, the proteins Unc-51 like autophagy activating kinase 1(ULK1), becline-1, autophagy related 1 protein (Atg4), and LC3 increased in the CRC cell lines HCT116 and SW480 in contrast to monotherapy with doxorubicin. The overexpression of these proteins indicated the beginning of autophagy flow through the activation of ULK1 and the hyperlipidation of LC3 at the beginning of this process. Moreover, we confirm that ULK1 is a bona fide target of hsa-miR-106a-5p (referred to from here on as miR-106a) in HCT116. We also observed through the GFP-LC3 fusion protein that in the presence of miR-106a, the accumulation of autophagy vesicles in cells stimulated with TT is inhibited. These results show that the TT triggered autophagy to modulate miR-106a/ULK1 expression, probably affecting different cellular pathways involved in cellular proliferation, survivance, metabolic maintenance, and cell death. Therefore, considering the importance of autophagy in cancer biology, the study of miRNAs that regulate autophagy in cancer will allow a better understanding of malignant tumors and lead to the development of new disease markers and therapeutic strategies.

Sesamin induces cell cycle arrest and apoptosis through p38/C-Jun N-terminal kinase mitogen-activated protein kinase pathways in human colorectal cancer cells.

Sesamin, a lignan compound, exhibits a variety of biological activities and possesses potent anticancer properties on some human cancers. However, its effect on human colorectal cancer (CRC) remains to be elucidated. To investigate the effects of sesamin on CRC cells and further to explore the mechanisms, cell viability, cell cycle and apoptosis assays were performed in this study. We found that sesamin had a selective antiproliferation of CRC cell line HCT116 in a dose- and time-dependent manner, but no obvious effect on human normal colorectal mucosa epithelial cell FHC. Further study showed that sesamin-induced cell cycle arrest and decreased the expression of Cyclin D1 significantly and dose-dependently in HCT116 cells. Moreover, sesamin dose-dependently triggered apoptosis of HCT116 but not FHC, and promoted the expression levels of proapoptotic biomarkers Bax, cleaved caspase-3 and cleaved PARP-1 and inhibited the expression of antiapoptotic biomarker Bcl-2. Western blot analysis was used to reveal the possible signaling pathways, and we found that sesamin upregulated the phosphorylation expression levels of C-Jun N-terminal kinase (JNK) and p38 except ERK1/2 in a dose-dependent way in both HCT116 and another CRC cell line SW480. Moreover, we found that the apoptosis effect induced by sesamin was partially eliminated by inhibiting JNK or p38 activation. Finally, we showed that sesamin effectively reduced the growth of xenograft tumors derived from cell lines with limited toxicity. Taken together, the potential ability of sesamin to induce cell cycle arrest and apoptosis was shown to be via the p38 and JNK mitogen-activated protein kinase signaling pathways, which may be one of the mechanisms of the anticancer activity of this low-toxic agent.

SIRT3-Mediated SOD2 and PGC-1α Contribute to Chemoresistance in Colorectal Cancer Cells.

Anticancer drugs generate excessive reactive oxygen species (ROS), which can cause cell death. Cancer cells can resist this oxidative stress, but the mechanism of resistance and associations with chemoresistance are unclear. Here, we focused on Sirtuin 3 (SIRT3), a deacetylating mitochondrial enzyme, in oxidative stress resistance in colorectal cancer (CRC). To evaluate SIRT3-related changes in mitochondrial function, ROS (mtROS) induction, and apoptosis, we used the human CRC cell lines HT29 and HCT116 transfected with short-hairpin RNA targeting SIRT3 and small interfering RNAs targeting superoxide dismutase 2 mitochondrial (SOD2) and peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α). In 142 clinical specimens from patients with CRC, we also assessed the association of SIRT3 protein levels (high/low) and prognosis. SIRT3 expression correlated with mtROS generation and apoptosis induction in cells treated with anticancer agents. Suppressing SIRT3 increased mtROS levels and cell sensitivity to anticancer agents. SIRT3 knockdown decreased SOD2 expression and activity, and suppressing SOD2 also improved sensitivity to anticancer drugs. In addition, SIRT3 was recruited with PGC-1α under oxidative stress, and suppressing SIRT3 decreased PGC-1α expression and mitochondrial function. PGC-1α knockdown decreased mitochondrial activity and increased apoptosis in cells treated with anticancer drugs. In resected CRC specimens, high vs low SIRT3 protein levels were associated with significantly reduced cancer-specific survival. SIRT3 expression affected CRC cell chemoresistance through SOD2 and PGC-1α regulation and was an independent prognostic factor in CRC. SIRT3 may be a novel target for CRC therapies and a predictive marker of sensitivity to chemotherapy.

Exosomal IDH1 increases the resistance of colorectal cancer cells to 5-Fluorouracil.

Chemoresistance challenges the clinical treatment of colorectal cancer and requires an urgent solution. Isocitrate dehydrogenase 1 (IDH1) is a key enzyme involved in glucose metabolism that mediates the malignant transformation of tumors. However, the mechanisms by which IDH1 is involved in colorectal cancer cell proliferation and drug resistance induction remain unclear. In this study, we found that IDH1 was highly expressed in human colorectal cancer tissues and could be used to indicate a high-grade tumor. In vitro gene overexpression and knockdown were used to determine whether IDH1 promoted the proliferation of the colorectal cancer cell line HCT8 and resistance to 5-Fluorouracil (5FU). Further studies have shown that the 5FU-resistant cell line, HCT8FU, secreted exosomes that contained a high level of IDH1 protein. The exosomal IDH1 derived from 5FU-resistant cells enhanced the resistance of 5FU-sensitive cells. Metabolic assays revealed that exosomes derived from 5FU-resistant cells promoted a decrease in the level of IDH1-mediated NADPH, which is associated with the development of 5FU resistance in colorectal cancer cells. Therefore, exosomal IDH1 may be the transmitter and driver of chemoresistance in colorectal cancer and a potential chemotherapy target.

An Efficient Synthesis of Novel Dextran-Arsenite Nanoparticles intended for Potential Antitumor Drug Material.

The functionalization of polysaccharides with synthetic nanopolymers has attracted great attention owing to the applications of this method in many industrial fields. This work aimed to investigate the effect of arsenic trioxide on the functionalization of dextran. Dextran-arsenite nanoparticle formation was induced by microwave with sulfuric acid as a catalyst. Various analytical techniques were used to verify the structure of the nanopolymers. Besides, various reaction conditions, such as dextran concentration, arsenic trioxide concentration and pH, were investigated to determine their impact on particle size. The results indicated that the product was an arsenite-based nanomaterial retaining the basic configuration of dextran and that the product size was positively correlated with pH but negatively correlated with arsenic trioxide concentration. Moreover, the inhibitory effects of the dextran-arsenite nanoparticles on the growth of the human colorectal cancer cell line HCT-116 and human hepatoma carcinoma cell lines Huh-7 and SMMC-7721 were studied. The results showed that the product could inhibit the proliferation of these three tumor cell lines in a dose-dependent manner. Therefore, the product could be a new type of functional nanomaterial for further study on the synthesis, biological activity and development of polysaccharide drugs.

Evaluation of cytotoxicity and anticancer activity of kombucha and doxorubicin combination therapy on colorectal cancer cell line HCT-116.

BACKGROUND:Colorectal cancer is the third leading to death type of cancer in the world. The therapeutic guideline varied between different methods. As the main therapeutic guideline is chemotherapy, recent studies had shown utilization of natural products in combination with conventional medication, elevate the efficiency of chemotherapeutic methods. Kombucha is a traditional beverage obtained from the fermentation of green tea as a rich source of flavonoid medicinal plant. This study aimed to evaluate the natural potential of combination therapy of this natural product with doxorubicin as a chemotherapeutic agent.MATERIALS AND METHODS:The study was performed as in vitro evaluation of biological activity of kombucha on HCT-116 cell line (human colon cancer cell line). The cytotoxic effect of different kombucha beverages (fermented green tea) in comparison with green tea extract was evaluated by dimethylthiazolyl tetrazolium bromide (MTT) assay. In the next step, anticancer activity of doxorubicin as a general guideline chemotherapeutic agent in combination with kombucha was evaluated by cell cycle analysis and apoptosis assay flow cytometry. Apoptotic genes expression pattern was determined using real-time polymerase chain reaction. The experiments were designed in three independent replications and statistically analyzed using SPSS software.RESULTS:The results show that kombucha compared with the green tea extract caused more (1.2 fold) early apoptosis induction and G0/G1 phase arrest. Moreover, kombucha increased the expression levels of p21, p53, and B-cell leukemia/lymphoma 2 (Bcl-2)-associated X protein genes (2, 2.5, and 1.5 fold, respectively) while it decreased Bcl-2 gene expression level (5–8 fold) compared with doxorubicin alone. Combination of kombucha with doxorubicin shows 2-fold increased G0/G1 phase compared with the doxorubicin treatment.CONCLUSION:This result indicated that kombucha caused boosted anticancer activity of doxorubicin agent. These findings suggest that kombucha may be has an assistor and useful role in colorectal cancer treatment align with chemotherapy.

Hypoxia facilitates the proliferation of colorectal cancer cells by inducing cancer-associated fibroblast-derived IL6

Colorectal cancer (CRC) is the most common malignancy worldwide, and its underlying molecular mechanisms remain largely unexplored. Accumulating evidences indicate cancer-associated fibroblasts (CAFs), abundant stromal cell population in the tumor microenvironment, play a key role in tumor development. Herein, we have successfully isolated CAFs and paired normal fibroblasts (NFs) from colorectal cancer tissues (n=10). By using a multiplex cytokine profiling assay, we have identified IL-6 as a major cytokine released by CAFs. Co-culturing of CAFs with CRC cell lines HCT116 or SW480 increases IL-6 release, and the secretion by CAFs can be further enhanced under hypoxia. By using the CCK-8 assay, we have found that HCT116 or SW480 cells treated with culture medium from CAFs, IL-6, or hypoxia showed a significant cell growth compared to control cells (p<0.01). Mechanistically, we have found that hypoxia could enhance the effect of the IL-6/STAT3 signaling on CRC cells, in part, through HIF-1a targeting PKM2. In conclusion, our data clearly proposes the interconnected mechanisms for constitutive activation of STAT3 signal by CAFs-derived IL-6 under hypoxia in colorectal cancer. The pharmacological inhibition of STAT3, PKM2, or HIF-1α can significantly reduce the oncogenic effect of IL-6, providing a potential therapeutic target for CRC patients.