Normal Colonic Epithelial Cells Research Articles

Circular RNA circWHSC1 facilitates colorectal cancer cell proliferation by targeting miR-130a-5p/zeb1 signaling in vitro and in vivo

Colorectal cancer is a prevalent cancer globally and has become a threaten of human health. Recently, circular RNAs (circRNAs) have been widely studied in the cancer area, and the function of circular RNA circWHSC1 has been identified in several cancers. However, the role of circWHSC1 in colorectal cancer remains elusive. In this study, we were interested in the effects of circWHSC1 on colorectal cancer progression. We found that level of circWHSC1 was elevated in colorectal cancer cells compared with normal colon epithelial cells. FISH assay further confirmed that circWHSC1 was mainly localized in cytoplasm. CircWHSC1 depletion repressed the viability of colorectal cancer cells. The colony formation number and Edu-positive colorectal cancer cells were inhibited by the depletion of circWHSC1, respectively. The knockdown of circWHSC1 promoted the apoptosis of colorectal cancer cells. The tumor growth of colorectal cancer cells in nude mice was attenuated by circWHSC1 silencing. Meanwhile, the invasion and migration ability of colorectal cancer cells was suppressed by circWHSC1 depletion. Mechanically, circWHSC1 targets miR-130a-5p to promote zeb1 expression in colorectal cancer cell. The depletion of circWHSC1 remarkably reduced the cell viability and Edu-positive colorectal cancer cells, and the miR-130a-5p inhibitor or zeb1 overexpression could restore the phenotypes. Furthermore, the tumor growth of colorectal cancer cells in nude mice was attenuated by circWHSC1 knockdown, while miR-130a-5p depletion or zeb1 overexpression reversed the effect in the model. Therefore, we concluded that Circular RNA circWHSC1 facilitated colorectal cancer cell proliferation by targeting miR-130a-5p/zeb1 signaling in vitro and in vivo.

Blockade of the SRC/STAT3/BCL-2 Signaling Axis Sustains the Cytotoxicity in Human Colorectal Cancer Cell Lines Induced by Dehydroxyhispolon Methyl Ether.

Colorectal cancer (CRC) is the third most prevalent human cancer globally. 5-Fluorouracil (5-FU)-based systemic chemotherapy is the primary strategy for advanced CRC treatment, yet is limited by poor response rate. Deregulated activation of signal transducer and activator of transcription 3 (STAT3) is fundamental to driving CRC malignant transformation and a poor prognostic marker for CRC, underscoring STAT3 as a promising CRC drug target. Dehydroxyhispolon methyl ether (DHME) is an analog of Hispolon, an anticancer polyphenol abundant in the medicinal mushroom Phellinus linteus. Previously, we have established DHME's cytotoxic effect on human CRC cell lines by eliciting apoptosis through the blockade of WNT/β-catenin signaling, a preeminent CRC oncogenic pathway. Herein, we unraveled that compared with 5-FU, DHME is a more potent killer of CRC cells while being much less toxic to normal colon epithelial cells. DHME suppressed both constitutive and interleukin 6 (IL-6)-induced STAT3 activation represented by tyrosine 705 phosphorylation of STAT3 (p-STAT3 (Y705)); notably, DHME-induced CRC apoptosis and clonogenicity limitation were abrogated by ectopic expression of STAT3-C, a dominant-active STAT3 mutant. Additionally, we proved that BCL-2 downregulation caused by DHME-mediated STAT3 blockage is responsible for DHME-induced CRC cell apoptosis. Lastly, DHME inhibited SRC activation, and v-src overexpression restored p-STAT3 (Y705) levels along with lowering the levels of apoptosis in DHME-treated CRC cells. We conclude DHME provokes CRC cell apoptosis by blocking the SRC/STAT3/BCL-2 axis besides thwarting WNT/β-catenin signaling. The notion that DHME targets two fundamental CRC signaling pathways underpins the potential of DHME as a CRC chemotherapy agent.

Crocin exerts anti-tumor effect in colon cancer cells via repressing the JAK pathway.

Crocin has been reported to have therapeutic effects on multiple cancers including colon cancer, but its specific mechanism is still ambiguous and needs to be further explored. Human colorectal adenocarcinoma cells (HCT-116) and human normal colonic epithelial cells (CCD841) were first treated with increasing concentrations of crocin. Subsequently, with 150 and 200 μM of crocin, the cell vitality was examined by cell counting kit 8. Cell apoptosis and proliferation were tested by TUNEL staining and colony formation assay, respectively. The expression of Ki-67 was assessed by immunofluorescence. Enzyme-linked immunosorbent assay was used to evaluate the level of inflammation- and oxidative-related factors. The reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) were examined by flow cytometer. Janus kinase (JAK), signal transducer and activator of transcription 3 (STAT3), and extracellular regulated protein kinases (ERK) in HCT-116 cells were tested by Western blot. Different concentrations of crocin barely affected the CCD841 cell vitality, while crocin restrained the HCT-116 cells vitality, proliferation and the expression of Ki-67, while inducing apoptosis in a concentration-dependent manner. Moreover, the contents of inflammation- and oxidative-related factors in HCT-116 cells were largely blunted by crocin that enhanced ROS and restrained the MMP and suppressed p-JAK2/JAK2, p-STAT3/STAT3, and p-ERK/ERK expression in HCT-116 cells. Crocin induced apoptosis and restored mitochondrial function in HCT-116 cells via repressing the JAK pathway. If the threptic effect works in patients, it could herald a new, effective treatment for colon cancer, improving the patients' prognosis and quality of life.

Exploring the Multifaceted Potential of a Peptide Fraction Derived from Saccharomyces cerevisiae Metabolism: Antimicrobial, Antioxidant, Antidiabetic, and Anti-Inflammatory Properties.

The rising demand for minimally processed, natural, and healthier food products has led to the search for alternative and multifunctional bioactive food components. Therefore, the present study focuses on the functional proprieties of a peptide fraction derived from Saccharomyces cerevisiae metabolism. The antimicrobial activity of the peptide fraction is evaluated against various foodborne pathogens, including Candida albicans, Candida krusei, Escherichia coli, Listeria monocytogenes, and Salmonella sp. The peptide fraction antioxidant properties are assessed using FRAP and DPPH scavenging capacity assays. Furthermore, the peptide fraction's cytotoxicity is evaluated in colorectal carcinoma and normal colon epithelial cells while its potential as an antidiabetic agent is investigated through α-amylase and α-glucosidase inhibitory assays. The results demonstrate that the 2-10 kDa peptide fraction exhibits antimicrobial effects against all tested microorganisms, except C. krusei. The minimal inhibitory concentration for E. coli, L. monocytogenes, and Salmonella sp. remains consistently low, at 0.25 mg/mL, while C. albicans requires a higher concentration of 1.0 mg/mL. Furthermore, the peptide fraction displays antioxidant activity, as evidenced by DPPH radical scavenging activity of 81.03%, and FRAP values of 1042.50 ± 32.5 µM TE/mL at 1.0 mg/mL. The peptide fraction exhibits no cytotoxicity in both tumor and non-tumoral human cells at a concentration up to 0.3 mg/mL. Moreover, the peptide fraction presents anti-inflammatory activity, significantly reducing the expression of the TNFα gene by more than 29.7% in non-stimulated colon cells and by 50% in lipopolysaccharide-stimulated colon cells. It also inhibits the activity of the carbohydrate digestive enzymes α-amylase (IC50 of 199.3 ± 0.9 µg/mL) and α-glucosidase (IC20 of 270.6 ± 6.0 µg/mL). Overall, the findings showed that the peptide fraction exhibits antibacterial, antioxidant, anti-inflammatory, and antidiabetic activity. This study represents a step forward in the evaluation of the functional biological properties of S. cerevisiae bioactive peptides.

Identification of the epigenetic mechanism for colorectal cancer stem cell regulation through the comprehensive microRNA expression profiling of human colorectal cancer tissues.

42 Background: MicroRNAs (miRNAs) are key regulators of cancer stem cell (CSC) functions, including self-renewal and differentiation. However, little is known about miRNAs that regulate such properties in human colorectal cancer. Methods: We compared the expression levels of 754 miRNAs between paired samples of EpCAM+/CD44+ cancer cells (enriched in CSCs) and EpCAM+/CD44neg cancer cells (non-tumorigenic cancer cells (NTCs)) sorted in parallel from human primary colorectal cancers by multiplex semi-quantitative PCR. Results: Comparison of the expression levels of miRNAs in the CSCs and NTCs resulted in the identification of 10 miRNAs upregulated (miR-221, miR-218, miR-500 and miR-195) or downregulated (miR-185, miR-19a, miR-337, miR-10a, miR-21 and miR-24) in CSCs compared to NTCs. Among them, miR-221 was most highly expressed in CSCs; and its expression level was very low in NTCs and normal colon epithelial cells (n=6, p<0.05). Kaplan-Myer estimates and univariate and multivariate analyses showed that higher miR-221 expression was an independent prognosis factor for poorer outcome. Constitutive overexpression of miR-221 enhanced organoid-forming capacity of both conventional colorectal carcinoma cell lines and patient-derived xenografts (PDX) in vitro. Importantly, constitutive downregulation of miR-221 suppressed organoid-forming capacity in vitro and substantially reduced the tumorigenic capacity of CSC populations from PDX lines in vivo. miR-221 directly targeted Quaking (QKI) 5, the most abundant splicing isoform of the human QKI gene, and suppressed its protein level. Knockdown of miR-221 suppressed the clonogenicity of colorectal CSCs in vitro in a QKI-expression dependent manner and ability to form tumors in vivo. Conclusions: Our study reveals a mechanistic link between miR-221 and QKI and highlights their key role in regulating CSC properties in human colorectal cancer and proposes that miR-221 will be a promising marker and a specific therapeutic target for human colorectal CSCs.

CIRC_0085323 SILENCING INHIBITS TNF-Α-INDUCED NORMAL HUMAN COLONIC EPITHELIAL CELL INFLAMMATION AND APOPTOSIS THROUGH THE MIR-495-3P/TRAF3 AXIS.

Background: Previous data have suggested the involvement of circular RNA (circRNA) in ulcerative colitis (UC) development. However, the role and mechanism of circ_0085323 in UC occurrence have not been reported. Methods: Normal human colonic epithelial cells (NCM460) were treated with TNF-α to simulate UC-like cell inflammation and injury in vitro. The expression of circ_0085323, microRNA-495-3p (miR-495-3p), and TNF receptor-associated factor 3 (TRAF3) was detected by quantitative real-time polymerase chain reaction. Protein expression was checked by western blotting analysis. Cell viability, cell proliferation, and cell apoptosis were investigated by cell counting kit-8 assay, 5-ethynyl-29-deoxyuridine assay, and flow cytometry analysis, respectively. IL-1β, IL-6, and IL-8 production were analyzed by enzyme-linked immunosorbent assays. Lactate dehydrogenase activity was assessed by a lactate dehydrogenase activity detection assay. The interactions among circ_0085323, miR-495-3p, and TRAF3 were identified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Results: Circ_0085323 was overexpressed in the colonic mucosal tissues of UC patients and TNF-α-stimulated NCM460 cells. Circ_0085323 knockdown relieved TNF-α-induced inhibitory effect on the proliferation of NCM460 cells and promoting effects on cell apoptosis and inflammation. Circ_0085323 acted as a miR-495-3p sponge, and the effects of circ_0085323 silencing on TNF-α-induced NCM460 cell injury were attenuated by decreasing miR-495-3p expression. TRAF3 was targeted by miR-495-3p, and circ_0085323 combined with miR-495-3p to regulate TRAF3. TRAF3 depletion not only alleviated TNF-α-induced NCM460 cell damage but also partially revoked the effect of circ_0085323 silencing combined with miR-495-3p depletion on TNF-α-induced NCM460 cell injury. Conclusions: Circ_0085323 knockdown ameliorated TNF-α-induced NCM460 cell injury by regulating the miR-495-3p/TRAF3 axis, which suggested that circ_0085323 might be a therapeutic target for UC.

New Schiff bases derived from dimethylpyridine-1,2,4-triazole hybrid as cytotoxic agents targeting gastrointestinal cancers: Design, synthesis, biological evaluation and molecular docking studies

In this research, a series of novel hybrid structures of dimethylpyridine-1,2,4-triazole Schiff bases were designed, synthesized, and evaluated for their in vitro cytotoxic potency on several human gastrointestinal cancer cells (EPG, Caco-2, LoVo, LoVo/Dx, HT29) and normal colonic epithelial cells (CCD 841 CoN). Schiff base 4h was the most potent compound against gastric EPG cancer cells (CC50 = 12.10 ± 3.10 μM), being 9- and 21-fold more cytotoxic than 5-FU and cisplatin, respectively. Moreover, it was not toxic to normal cells. Regarding the cytotoxicity against colorectal cancer cells, compounds 4d and 4l exhibited good activity against HT29 cells (CC50 = 52.80 ± 2.80 μM and 61.40 ± 10.70 μM, respectively), and were comparable to or more potent than cisplatin and 5-FU. Also, they were less toxic to normal cells with a higher selectivity index (SI, CCD 841 CoN/HT29 = 4.20 and 2.85, respectively) than reference drugs (SI, CCD 841 CoN/HT29 < 1). Selected Schiff bases were subjected to the P-glycoprotein inhibition assay. Schiff bases 4d, 4e, and 4l influenced P-gp efflux function, significantly increasing the accumulation of rhodamine 123 in colon cancer cell lines. Further mechanistic studies showed that compound 4l induced apoptotic cell death through a caspase-dependent mechanism and by regulating the p53-MDM2 signaling pathway in HT29 cells. Also, physicochemical predictions of compounds 4d, 4e, 4h, and 4i were examined in silico. The results revealed that the compounds possessed promising drug-likeness profiles.

Probiotics ameliorate benzene-induced systemic inflammation and hematopoietic toxicity by inhibiting Bacteroidaceae-mediated ferroptosis

The intestinal microbiota is associated with the development of benzene-induced hematopoietic toxicity. Modulation of intestinal homeostasis by probiotic supplementation has been considered an effective strategy to prevent adverse health effects. However, the role and mechanism of probiotics in benzene-induced hematopoietic toxicity are unclear. After 45 days of exposure, benzene caused bone marrow hematopoietic toxicity in mice. Furthermore, we found that benzene altered the intestinal barrier in mice, leading to an increase in the abundance of Bacteroidaceae and the activation of systemic inflammation. Interestingly, Fe2+ accumulation, lipid peroxidation, and differential expression of ferroptosis proteins were observed in the intestinal tissues of benzene-exposed mice. After fecal microbiota transplantation, stool microbes from benzene-exposed mice led to the development of intestinal ferroptosis in recipient mice. In particular, oral probiotics significantly reversed elevated Bacteroidaceae and intestinal ferroptosis, ultimately improving benzene-induced hematopoietic damage. We further used the benzene metabolite 1,4-BQ to treat human normal colonic epithelial cells (NCM460) and intervened with the ferroptosis inhibitor liproxstatin-1 (Lip-1) to validate the relationship between intestinal ferroptosis and inflammation. The results showed that 1,4-BQ treatment resulted in increased intracellular ROS levels and abnormal expression of ferroptosis proteins and the inflammatory factors IL-5 and IL-13. However, the use of Lip-1 significantly inhibited oxidative stress, ferroptosis, and inflammation in NCM460 cells. This result suggested that ferroptosis might be involved in benzene-induced hematopoietic toxicity by mediating Th2-type systemic inflammation. Overall, these findings revealed a role for Bacteroidaceae-intestinal ferroptosis-inflammation in benzene-induced hematopoietic toxicity and highlighted that probiotics could be a promising strategy to prevent adverse hematologic outcomes.

Divergent roles of estrogen receptor subtypes in regulating estrogen-modulated colonic ion transports and epithelial repair

Although it was described previously for estrogen (E2) regulation of intestinal epithelial Cl- and HCO3- secretion in sex difference, almost nothing is known about the roles of estrogen receptor (ER) subtypes in regulating E2-modulated epithelial ion transports and epithelial restitution. Here, we aimed to investigate ERα and ERβ subtypes in the regulation of E2-modulated colonic epithelial HCO3- and Cl- secretion and epithelial restitution. Through physiological and biochemical studies, in combination of genetic knockdown, we showed that ERα attenuated female colonic Cl- secretion but promoted Ca2+-dependent HCO3- secretion via store-operated calcium entry (SOCE) mechanism in mice. However, ERβ attenuated HCO3- secretion by inhibiting Ca2+via the SOCE and inhibiting cAMP via protein kinases. Moreover, ERα but not ERβ promoted epithelial cell restitution via SOCE/Ca2+ signaling. ERα also enhanced cyclin D1, proliferating cell nuclear antigen, and β-catenin expression in normal human colonic epithelial cells. All ERα-mediated biological effects could be attenuated by its selective antagonist and genetic knockdown. Finally, both ERα and ERβ were expressed in human colonic epithelial cells and mouse colonic tissues. We therefore conclude that E2 modulates complex colonic epithelial HCO3- and Cl- secretion via ER subtype-dependent mechanisms and that ERα is specifically responsible for colonic epithelial regeneration. This study provides novel insights into the molecular mechanisms of how ERα and ERβ subtypes orchestrate functional homeostasis of normal colonic epithelial cells.

Scutellaria baicalensis enhances 5-fluorouracil-based chemotherapy via inhibition of proliferative signaling pathways

Fluoropyridine-based chemotherapy remains the most widely used treatment for colorectal cancer (CRC). In this study, we investigated the mechanism by which the natural product Scutellaria baicalensis (Huang Qin; HQ) and one of its main components baicalin enhanced 5-fluorouracil (5-FU) antitumor activity against CRC. Cell proliferation assays, cell cycle analysis, reverse-phase protein array (RPPA) analysis, immunoblot analysis, and qRT-PCR were performed to investigate the mechanism(s) of action of HQ and its active components on growth of CRC cells. HQ exhibited in vitro antiproliferative activity against drug resistant human CRC cells, against human and mouse CRC cells with different genetic backgrounds and normal human colon epithelial cells. In vivo animal models were used to document the antitumor activity of HQ and baicalin. The mechanism of growth inhibitory activity of HQ is due to inhibition of proliferative signaling pathways including the CDK-RB pathway. In addition, HQ enhanced the antitumor effects of 5-FU and capecitabine in vivo. Furthermore, we identified baicalin as an active component of HQ. The combination of baicalin and 5-FU demonstrated synergistic activity against 5-FU-resistant RKO-R10 cells. The combination significantly inhibited in vivo tumor growth greater than each treatment alone. RPPA results showed that the signaling pathway alterations in CRC cells were similar following HQ and baicalin treatment. Together, these results indicate that HQ and its component baicalin enhance the effect of 5-fluorouracil-based chemotherapy via inhibition of CDK-RB pathway. These findings may provide the rational basis for developing agents that can overcome the development of cellular drug resistance.9Q3bw5kPrTjBvtYuEHSNmaVideo

Insights into Q-markers and molecular mechanism of Sanguisorba saponins in treating ulcerative colitis based on lipid metabolism regulation

BackgroundSanguisorba saponin extract (SSE) is the main active part of Sanguisorba officinalis with various pharmacological activities such as anti-inflammatory, anti-bacterial and anti-oxidant. However, its therapeutic role and underlying mechanisms for ulcerative colitis (UC) still need to be elucidated. PurposeThis study aims to explore the therapeutic effect, effectiveness-material basis-quality markers (Q-markers) and prospective mechanism of function of SSE on UC. MethodsFresh 2.5% dextran sulfate sodium salt (DSS) solution was placed in drinking bottles for 7 days to induce a mouse model of UC. SSE and sulfasalazine (SASP) were supplemented to mice by gavage for consecutive 7 days to investigate the therapeutic role of SSE on UC. Mouse monocyte macrophages (RAW264.7) and human normal colonic epithelial (NCM460) cells were treated with LPS to induce inflammatory responses, followed by pharmacodynamic examination with different concentrations of SSE. Hematoxylin-eosin (HE) and Alcian blue staining were conducted to evaluate the pathological damage of mice colon. Lipidomic technology was conducted to explore the differential lipids closely related to the disease process of UC. Quantitative PCR analysis, immunohistochemistry and ELISA kit were used to measure the expression levels of the corresponding proteins and pro-inflammatory factors. ResultsSSE treatment could effectively reduce the elevated expressions of pro-inflammatory factors in RAW264.7 and NCM460 cells due to LPS stimulation. Intragastric administration of SSE was found to significantly alleviate the symptoms of DSS-induced colon injury and low-polar saponins in SSE. Low polarity saponins, especially ZYS-II, were proved to be the main active substances of SSE in treating UC. In addition, SSE could significantly ameliorate the aberrant lipid metabolism in UC mice. The role of phosphatidylcholine (PC)34:1 in the UC pathogenesis has been fully verified in our previous studies. Herein, SSE-dosing effectively reversed the metabolic disorder of PCs in UC mice, and increased the PC34:1 level to normal via up-regulating the expression of phosphocholine cytidylyltransferase (PCYT1α). ConclusionOur data innovatively revealed that SSE could significantly alleviate the symptoms of UC by reversing the disorder of PC metabolism induced by DSS modeling. SSE was proved for the first time to be a promising and effective candidate for UC treatment.

Protective Effects of Piceatannol on DNA Damage in Benzo[a]pyrene-Induced Human Colon Epithelial Cells

Evidence shows that the dietary intake of polycyclic aromatic hydrocarbons (PAHs) from food processing induces the cellular DNA damage response and leads to the development of colorectal cancer (CRC). Therefore, protecting from cellular DNA damage might be an effective strategy in preventing CRC. Benzo[a]pyrene (B[a]P) was used as a CRC initiator in the present study. Compared with other stilbenoids, piceatannol (PIC) showed the most effective inhibition of B[a]P-induced cytochrome P450 1B1 (CYP1B1) protein expression in NCM460 normal human colon epithelial cells. PIC treatment alleviated DNA migration and enhanced the expression of DNA-repair-related proteins, including histone 2AX (H2AX), checkpoint kinase 1 (Chk1), and p53, in B[a]P-induced NCM460 cells. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA) revealed that PIC exerted antioxidative effects on NCM460 cells by increasing the glutathione (GSH) content and scavenging the excess intracellular reactive oxygen species (ROS) induced by B[a]P. Furthermore, PIC suppressed B[a]P-induced CYP1B1 protein expression and stimulated miR-27b-3p expression. The upregulation of phase II detoxification enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH) and quinone oxidoreductase 1 (NQO1), and the antioxidative enzyme, heme oxygenase 1 (HO-1), via the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway was observed in the PIC-treated group. Our results suggest that PIC is a potential CRC-blocking agent due to its ability to alleviate DNA damage, decrease intracellular ROS production, modulate the metabolism and detoxification of B[a]P, and activate the Nrf2 signaling pathway in B[a]P-induced NCM460 cells.

MTOR inhibition suppresses Myc-driven polyposis by inducing immunogenic cell death

Myc is a key driver of colorectal cancer initiation and progression, but remains a difficult drug target. In this study, we show that mTOR inhibition potently suppresses intestinal polyp formation, regresses established polyps, and prolongs lifespan of APCMin/+ mice. Everolimus in diet strongly reduces p-4EBP1, p-S6, and Myc levels, and induces apoptosis of cells with activated β-catenin (p-S552) in the polyps on day 3. The cell death is accompanied by ER stress, activation of the extrinsic apoptotic pathway, innate immune cell recruitment, and followed by T-cell infiltration on day 14 persisting for months thereafter. These effects are absent in normal intestinal crypts with physiologic levels of Myc and a high rate of proliferation. Using normal human colonic epithelial cells, EIF4E S209A knockin and BID knockout mice, we found that local inflammation and antitumor efficacy of Everolimus requires Myc-dependent induction of ER stress and apoptosis. These findings demonstrate mTOR and deregulated Myc as a selective vulnerability of mutant APC-driven intestinal tumorigenesis, whose inhibition disrupts metabolic and immune adaptation and reactivates immune surveillance necessary for long-term tumor control.

Discovery of novel benzamide derivatives bearing benzamidophenyl and phenylacetamidophenyl scaffolds as potential antitumor agents via targeting PARP-1.

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a crucial role in DNA damage repair and has been identified as a promising therapeutic target in cancer therapy. As a continuation of our efforts on the development of novel PARP-1 inhibitors with potent anticancer activity, a series of benzamide derivatives containing the benzamidophenyl and phenylacetamidophenyl scaffolds were designed and synthesized based on the structure optimization of our previously reported compound IX. All target compounds were screened for their in vitro antiproliferative activities against human colorectal cancer cells (HCT116, DLD-1 and SW480) and human normal colonic epithelial cells (NCM460). Among them, compound 13f exhibited the most potent anticancer activity against HCT116cells and DLD-1cells with IC50=0.30μM and 2.83μM, respectively. Moreover, 13f displayed significant selectivity in inhibiting HCT116 cancer cells over the normal NCM460cells. Furthermore, 13f exhibited excellent PARP-1 inhibitory effect with IC50=0.25nM. Besides, 13f was found to effectively inhibit colony formation and migration of HCT116cells. Studies on the mechanisms revealed that 13f could arrest cell cycle at G2/M phase, accumulate DNA double-strand breaks, reduce mitochondrial membrane potential and ultimately induce apoptosis in HCT116cells. In addition, molecular docking study indicated that 13f could combine firmly with the catalytic pocket of PARP-1 through multiple hydrogen bond interactions. Collectively, these findings demonstrated that 13f could serve as a promising anticancer candidate and deserves further investigation.

Quassinoid analogs exert potent antitumor activity via reversible protein biosynthesis inhibition in human colorectal cancer

Cellular protein synthesis is accelerated in human colorectal cancer (CRC), and high expression of protein synthesis regulators in CRC patients is associated with poor prognosis. Thus, inhibition of protein synthesis may be an effective therapeutic strategy for CRC. We previously demonstrated that the quassinoid bruceantinol (BOL) had antitumor activity against CRC. Herein, potent tumor growth suppression (>80%) and STAT3 inhibition was observed in two different mouse models following BOL administration. Loss of body and spleen weight was observed but was eliminated upon nanoparticle encapsulation while maintaining strong antitumor activity. STAT3 siRNA knockdown exhibited modest suppression of cell proliferation. Surprisingly, STAT3 inhibition using a PROTAC degrader (SD-36) had little effect on cancer cell proliferation suggesting the possibility of additional mechanism(s) of action for quassinoids. BOL-resistant (BR) cell lines, HCT116BR and HCA7BR, were equally sensitive to standard CRC therapeutic agents and known STAT3 inhibitors but resistant to homoharringtonine (HHT), a known protein synthesis inhibitor. The ability of quassinoids to inhibit protein synthesis was dependent on the structure of the C15 sidechain. Of note, BOL did not inhibit protein synthesis in normal human colon epithelial cells whereas HHT and napabucasin remained effective in these normal cells. Novel quassinoids were designed, synthesized, and evaluated in pre-clinical CRC models. Treatment with the most potent analog, 5c, resulted in significant inhibition of cell proliferation and protein synthesis at nanomolar concentrations. These quassinoid analogs may represent a novel class of protein synthesis inhibitors for the treatment of human CRC.

Proliferation Inhibitory Activity of Quinones from Blaps rynchopetera Defense Secretion on Colorectal Tumor Cells.

To explore the proliferation inhibitory effect of quinones from Blaps rynchopetera defense secretion on colorectal tumor cell lines. Human colorectal cancer cell HT-29, human colorectal adenocarcinoma cell Caco-2 and normal human colon epithelial cell CCD841 were chosen for the evaluation of inhibitory activity of the main quinones of B. rynchopetera defense secretion, including methyl p-benzoquinone (MBQ), ethyl p-benzoquinone (EBQ), and methyl hydroquinone (MHQ), through methyl thiazolyl tetrazolium assay. The tumor-related factors, cell cycles, related gene expressions and protein levels were detected by enzyme-linked immunosorbent assy, flow cytometry, RT-polymerase chain reaction and Western blot, respectively. MBQ, EBQ, and MHQ could significantly inhibit the proliferation of Caco-2, with half maximal inhibitory concentration (IC50) values of 7.04 ± 0.88, 10.92 ± 0.32, 9.35 ± 0.83, HT-29, with IC50 values of 14.90 ± 2.71, 20.50 ± 6.37, 13.90 ± 1.30, and CCD841, with IC50 values of 11.40 ± 0.68, 7.02 ± 0.44 and 7.83 ± 0.05 µg/mL, respectively. Tested quinones can reduce the expression of tumor-related factors tumor necrosis factor α, interleukin (IL)-10, and IL-6 in HT-29 cells, selectively promote apoptosis, and regulate the cell cycle which can reduce the proportion of cells in the G1 phase and increase the proportion of the S phase. Meanwhile, tested quinones could up-regulate mRNA and protein expression of GSK-3β and APC, while down-regulate that of β-catenin, Frizzled1, c-Myc, and CyclinD1 in the Wnt/β-catenin pathway of HT-29 cells. Quinones from B. rynchopetera defense secretion could inhibit the proliferation of colorectal tumor cells and reduce the expression of related factors, which would be functioned by regulating cell cycle, selectively promoting apoptosis, and affecting Wnt/β-catenin pathway-related mRNA and protein expressions.

Paris polyphylla Sm. Induces Reactive Oxygen Species and Caspase 3-Mediated Apoptosis in Colorectal Cancer Cells In Vitro and Potentiates the Therapeutic Significance of Fluorouracil and Cisplatin.

Paris polyphylla Sm. (Melanthiaceae) is an essential, vulnerable herb with a wide range of traditional applications ranging from fever to cancer in various communities. The use of P. polyphylla in India is limited to traditional healers. Here, we demonstrated that P. polyphylla extract (PPE) has good phenol, flavonoid, saponin, and steroidal saponin content and anti-oxidant activity with IC50 35.12 ± 6.1 μg/mL in DPPH and 19.69 ± 6.7 μg/mL in ABTS. Furthermore, PPE induces cytotoxicity in HCT-116 with IC50 8.72 ± 0.71 μg/mL without significant cytotoxicity inthe normal human colon epithelial cell line, CCD 841 CoN. PPE inhibits the metastatic property and induces apoptosis in HCT-116, as measured by Annexin V/PI, by increasing the production of reactive oxygen species (ROS) and caspase 3 activation. PPE acts synergistically with 5FU and cisplatin in HCT-116 and potentiates their therapeutic significance. Steroidal saponins with anticancer activities were detected in PPE by HR-LCMS. The present study demonstrated that PPE induces apoptosis by increasing ROS and activating caspase 3, which was attributed to steroidal saponins. PPE can be used as a potential natural remedy for colon cancer.

Cellular Localization of Selected Porphyrins and Their Effect on the In Vitro Motility of Human Colon Tumors and Normal Cells.

Standard therapies for colorectal cancer cannot eliminate or sufficiently reduce the metastasis process. Photodynamic therapy (PDT) may be an alternative to minimizing this problem. Here, we examined the cellular localization of selected porphyrins and determined whether free-base and manganese (III) metallated porphyrins may limit colon cancer cells' (HT29) or normal colon epithelial cells' (CCD 841 CoTr) motility in vitro. White light irradiation was used to initiate the photodynamic effect. Porphyrin uptake by the cells was determined by porphyrin fluorescence measurements through the use of confocal microscopy. Free-base porphyrin was found in cells, where it initially localized at the edge of the cytoplasm and later in the perinuclear area. The concentrations of porphyrins had no effect on cancer cell migration but had a significant effect on normal cell motility. Due to the low concentrations of porphyrins used, no changes in F-actin filaments of the cellular cytoskeleton were detected. Signal transmission via connexons between neighbouring cells was limited to a maximum of 40 µm for HT29 and 30 µm for CCD 841 CoTr cells. The tested porphyrins differed in their activity against the tumor and normal cells' migration capacity. Depending on the porphyrin used and the type of cells, their migration changed in relation to the control sample. The use of white light may change the activity of the porphyrins relative to the migratory capacity of the cells. The aim of the present study was to analyse the intracellular localization of tested porphyrins and their influence on the mobility of cells after irradiation with harmless white light.

High-throughput isolation of gentiopicroside from Gentiana lutea and the study on its anticancer properties in vitro

The study aims at the isolation of gentiopicroside (GPC) from Gentiana lutea L. root extracts. GPC is a bioactive glycosylated secoiridoid from Gentiana spp. (Gentianaceae) proved to exhibit broad pharmaceutical activities, including anti-inflammatory, hepatoprotective and spasmolytic action. Due to its marked potential, an efficient recovery method of high purity GPC from G. lutea roots was elaborated using the combination of Centrifugal Partition Extraction (CPE) that offers the advantage of industrial scalability, and Preparative High Performance Liquid Chromatography (prep-HPLC) techniques. For this purpose the root hydroalcoholic extract of G. lutea was initially fractionated in a 0.3 L volume CPE column using the biphasic solvent system that was composed of heptane: ethyl acetate: ethanol: water 1:8:3:5 (v/v/v/v) in elution-extrusion mode. As a result, 2.2 g of GPC (purity > 90%) and 0.77 g of GPC-enriched fractions (purity > 80%) were obtained from 31.5 g of the extract within 6 h (5 consecutive CPE fractionations). The following prep-HPLC based purification step increased the quantity and purity of the finally isolated secoiridoid to 2.21 g of > 96% pure GPC. The successful recovery of large amounts of pure GPC enabled the study on the anti-proliferative activity against the following cancer and normal cell lines: lung (A549, NCI-H1299), breast (MCF7, MDA-MB-468), glioblastoma (T98G), rhabdomyosarcoma (TE671), colon (HT-29, DLD-1) cancer cells and normal colon epithelial cells (CCD 841 CoTr). The MTT assay enabled the evaluation of the dose-dependent growth inhibition effect that was evident in all analyzed cancer cell lines, however, TE671 cells were the most sensitive (IC50 = 0.078 mg/mL) and MDA-MB-468 cells were the least sensitive (IC50 = 0.208 mg/mL) to the GPC treatment. The tested compound showed only a weak cytotoxic effect against normal cells (CCD 841 CoTr).

Prevention of Cell Proliferation Signaling Via RNA Interference of c-myc Gene Expression in Colon Cancer In-vitro

Colon cancer is a cancer begins in the large intestine (colon). Its aggression is due to late diagnosis, so poor prognosis and higher mortality rates are reported. Colon cancer has become a thorny research area that requires more examination of cellular pathways involved in its emergency. Based on this, we sought to check the biological role of cMyc in colon cancer cell proliferation through targeting its expression by a respective siRNA using CaCo-2 cell lines. Notably, the cytotoxic potential of cMyc knockdown was monitored by cell imaging using an inverted microscope, the production of lactate dehydrogenase (LDH) and viability of the transfected cells using MTT assay. The relative gene expression of c-myc and other related factors such as Raf-1 was investigated using quantitative real-time PCR (qRT-PCR). ELISA assay has been used to achieve the production of pro and anti-inflammatory cytokines released from transfected cells. Interestingly, the transfection with a new designed siRNA antagonist cMyc markedly decreased CaCo-2 cell viability rate in a dose-dependent manner compared with the transfection with specific siRNA antagonist Luciferase gene, which served as control. Likewise, number of survived cells significantly reduced following transfection with siRNA against c-myc, while the relative production of LDH significantly increased. Importantly, the cytotoxic effect of the transfection protocol showed neglected influence in normal colon epithelial cells (NCM-460 cell lines) indicating the selective regulation of colon cancer cell proliferation by transfection with siRNA targeting c-myc. Noteworthy, the knockdown efficiency of c-myc showed more than 80% inhibition of its expression in CaCo-2 cells transfected with the siRNA antagonist c-myc indicated by qRT-PCR. The relative gene expression of Raf-1 significantly decreased in c-myc knockdown cells, while the relative expression of both P53 and Caspase 3, as cell death indicators, significantly upregulated compared with control-transfected cells. Furthermore, the transfection with siRNA targeting c-myc markedly increased the production of interleukin 1 alpha (IL-1α ) and IL-1β, as pro-inflammatory cytokines, while decreased the production of IL- 4 and IL-10, as anti-inflammatory cytokines. These data provide evidence for the involvement of c-myc gene expression in colon cancer cell proliferation and immune response during cancer development.