Inhibits Colon Cancer Cell Growth Research Articles

Ginseng berry concentrate prevents colon cancer via cell cycle, apoptosis regulation, and inflammation-linked Th17 cell differentiation.

The Asian ginseng root (Panax ginseng C.A. Meyer) is a very commonly used herbal medicine worldwide. Ginseng fruit, including the berry (or pulp) and seed, is also valuable for several health conditions including immunostimulation and cancer chemoprevention. In this study, the anticancer and anti-proliferative effects of the extracts of ginseng berry and seed were evaluated. The ginsenosides in the ginseng berry concentrate (GBC) and ginseng seed extract (GSE) were analyzed. We then evaluated their anti-colorectal cancer potentials, including antiproliferation, cell cycle arrest, and apoptotic induction. Further investigation consisted of the berry's adaptive immune responses, such as the actions on the differentiation of T helper cells Treg, Th1, and Th17. The major constituents in GBC were ginsenosides Re and Rd, which can be compared to those in the root. The GBC significantly inhibited colon cancer cell growth, and its anti-proliferative effect involved mechanisms including G2/M cell cycle arrest via upregulation of cyclin A and induction of apoptosis via regulation of apoptotic related gene expressions. GBC also downregulated the expressions of pro-inflammatory cytokine genes. For the adaptive immune responses, GBC did not influence Th1 and Treg cell differentiation but significantly inhibited Th17 cell differentiation and thus regulated the balance of Th17/Treg for adaptive immunity. Although no ginsenoside was detected in the GSE, interestingly, it obviously enhanced colon cancer cell proliferation with the underlined details to be determined. Our results suggested that GBC is a promising dietary supplement for cancer chemoprevention and immunomodulation.

Novel lncRNA LINC00941 Promotes Proliferation and Invasion of Colon Cancer Through Activation of MYC.

PurposeWe conducted the study to elucidate how LncRNA LINC00941 affects colon cancer progression and its possible regulatory mechanism.MethodsThe expression level of LINC00941 in colon cancer tissues and cells was detected by qRT-PCR. The function of LINC00941 on colon cancer cell proliferation, migration, and invasion was detected by CCK-8 and Transwell assay respectively. The target interactions among LINC00941, miR-205-5p, and MYC were further confirmed by dual-luciferase reporter gene assays and RNA pull-down experiments. Meanwhile, in vivo experiments were carried out to study the role of LINC00941 in the xenotransplantation model.ResultsLINC00941 expression level was elevated in colon cancer tissues and cells. LINC00941 overexpression accelerated proliferation, migration, and invasion of colon cancer cells, while the LINC00941 knockdown showed the opposite results. In addition, LINC00941 regulated the expression of MYC by sponging miR-205-5p as a competitive endogenous RNA, and miR-205-5p knockdown reversed the tumor inhibition of LINC00941 knockdown on colon cancer cells. Xenograft model assay confirmed that LINC00941 silencing could inhibit colon cancer cell growth and metastasis.ConclusionLINC00941 may markedly promote colon cancer progression by acting on the miR-205-5p/MYC axis as a ceRNA, which offers novel clues for lncRNA to guide the treatment and prognosis of colon cancer.

Biogenic selenium nanoparticles produced by Lactobacillus casei ATCC 393 inhibit colon cancer cell growth in vitro and in vivo

Selenium compounds exhibit excellent anticancer properties but have a narrow therapeutic window. Selenium nanoparticles, however, are less toxic compared to other selenium forms, and their biogenic production leads to improved bioavailability. Herein, we used the probiotic strain Lactobacillus casei ATCC 393, previously shown to inhibit colon cancer cell growth, to synthesize biogenic selenium nanoparticles. We examined the anticancer activity of orally administered L. casei, L. casei-derived selenium nanoparticles and selenium nanoparticle-enriched L. casei, and investigated their antitumor potential in the CT26 syngeneic colorectal cancer model in BALB/c mice. Our results indicate that L. casei-derived selenium nanoparticles and selenium nanoparticle-enriched L. casei exert cancer-specific antiproliferative activity in vitro. Moreover, the nanoparticles were found to induce apoptosis and elevate reactive oxygen species levels in cancer cells. It is noteworthy that, when administered orally, selenium nanoparticle-enriched L. casei attenuated the growth of colon carcinoma in mice more effectively than the isolated nanoparticles or L. casei, suggesting a potential additive effect of the nanoparticles and the probiotic. To the best of our knowledge this is the first comparative study examining the anticancer effects of selenium nanoparticles synthesized by a microorganism, the selenium nanoparticle-enriched microorganism and the sole microorganism.

The therapeutic effect of the BRD4-degrading PROTAC A1874 in human colon cancer cells

A1874 is a novel BRD4-degrading proteolysis targeting chimera (PROTAC). In primary colon cancer cells and established HCT116 cells, A1874 potently inhibited cell viability, proliferation, cell cycle progression, as well as cell migration and invasion. The BRD4-degrading PROTAC was able to induce caspase and apoptosis activation in colon cancer cells. Furthermore, A1874-induced degradation of BRD4 protein and downregulated BRD-dependent genes (c-Myc, Bcl-2, and cyclin D1) in colon cancer cells. Significantly, A1874-induced anti-colon cancer cell activity was more potent than the known BRD4 inhibitors (JQ1, CPI203, and I-BET151). In BRD4-knockout colon cancer cells A1874 remained cytotoxic, indicating the existence of BRD4-independent mechanisms. In addition to BRD4 degradation, A1874 cytotoxicity in colon cancer cells was also associated with p53 protein stabilization and reactive oxygen species production. Importantly, the antioxidant N-acetyl-cysteine and the p53 inhibitor pifithrin-α attenuated A1874-induced cell death and apoptosis in colon cancer cells. In vivo, A1874 oral administration potently inhibited colon cancer xenograft growth in severe combined immuno-deficient mice. BRD4 degradation and p53 protein elevation, as well as apoptosis induction and oxidative stress were detected in A1874-treated colon cancer tissues. Together, A1874 inhibits colon cancer cell growth through both BRD4-dependent and -independent mechanisms.

Abstract 3999: Modeling the tumor microenvironment architecture of metastatic colorectal cancer in 3D organoids

Abstract A tumor mass does not consist of merely cancerous cells but also the tightly surrounding tumor microenvironment (TME), made up of both the non-cellular extracellular matrix (ECM) and various types of stromal cells. Though chemotherapy resistance is often attributed to tumor heterogeneity, cancer progression and malignancy is also profoundly influenced by its interaction with the surrounding TME. Residing hepatic stellate cells (HSC) in the liver are considered a major component of a liver TME because they transdifferentiate into highly proliferative and motile myofibroblasts that are associated with desmoplasia and tumor growth. HSC activation can occur in response to various cytokines, such as TGF-ß, from either a neighboring cell in response to an injury or from cancerous cells within the tissue. Myofibroblastic HSCs are responsible for the major changes that occur in the liver ECM associated with an aggressive TME via two mechanisms, the deposition of new ECM components and the physical remodeling of the preexisting ECM. A healthy, balanced TME is thought to result in a less aggressive, more vulnerable cancer while a more remodeled, unbalanced TME that consists of activated HSCs result in a more aggressive and less responsive cancer to chemotherapy. In order to test this relationship between the TME architecture and cancer cell malignancy as well as chemoresistance, we have developed a 3-dimensional organoid model that can assess the effect of a remodeled TME on a cancer spheroid. Our lab has published the general organoid system that we utilize here; in short, we embedded a colorectal cancer cell line (HCT116) spheroid into an organoid consisting of HSCs (LX2) and collagen type 1. We then activated the LX2s to become myofibroblastic with exogenous TGF-ß causing excessive deposition and remodeling of collagen around the cancer spheroid. The remodeled TME that resulted from TGF-ß-mediated activation was quantified by analyzing the stiffness of the organoid that surrounds the spheroid as well as the in-depth, computational image analysis of the collagen fibers via CT-FIRE. Our compression data, measured rheologically, revealed TGF-ß-mediated activation of the HSCs resulted in a stiffening of the organoid around the tumor spheroid. Results from the Picrosirus Red staining revealed the more remodeled organoid, containing activated HSCs, produced longer, wider, and more aligned characteristics around the spheroid. Our immunohistochemistry data indicated that the remodeled TME inhibits colon cancer cell growth and migration within the organoid. This inhibited proliferation, in combination with the physical collagen barrier in the activated TME, is thought to have a large effect on the ability for a chemotherapy to successfully penetrate and induce cytotoxic effects on the cancer cells. Collectively, the ability to manipulate the tumor organoids' TME towards fibrosis enables us to predict chemotherapy response based on TME properties and not solely cancer cells in isolation. Citation Format: Anthony Dominijanni. Modeling the tumor microenvironment architecture of metastatic colorectal cancer in 3D organoids [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3999.

Abstract 508: A novel orally available Wnt-pathway inhibitor for the treatment of metastatic cancers

Abstract We have developed a novel drug compound (OBD9) which blocks growth of metastatic cancer cells both in vitro and in vivo. We now identify OBD9 as an effective Wnt-signaling inhibitor targeting TNIK (TRAF2 And NCK Interacting Kinase) and we hypothesize that OBD9 represent a novel therapeutic option for patients with tumors that have an activated Wnt signaling pathway. To find novel drug candidates that selectively inhibit metastatic tumor cell viability, a drug screen was firstly performed and results of the screen identified members of the FDA-approved benzimidazole methylcarbamate family (e.g. mebendazole (MBZ) and albendazole (ALB)) as potential therapies for metastatic cancer. Earlier work also supports a role for this chemical family in the potential treatment of multiple cancers, but progress has been stalled by their poor water solubility and poor bioavailability for systemic delivery to disseminated tumors. We therefore synthesized a novel compound (OBD9) containing the scaffold of MBZ coupled to an oxetane group to enhance aqueous solubility to 361μM. OBD9 demonstrates significant cytotoxicity toward a variety of cancer cell types including colon, lung, and prostate cancers (IC50: 0.9-2μM). In a mouse xenograft model using highly aggressive PCMLN3 prostate cancer cells, OBD9 at 30 mg/kg significantly repressed growth of established tumors with no visible toxicity. In a mouse xenograft model of human A549 lung cancer cell line, orally delivered OBD9 also dramatically inhibited the growth of established tumors at 30 or 90μM without noticeable toxicity. Mechanistically, we find that OBD9 treatment significantly reduces TNIK levels as early as 4 hours at 1uM via an autophagy-dependent protein degradation pathway. TNIK functions as an activator of Wnt signaling pathway via phosphorylation of the beta-catenin/TCF4 complex that regulates Wnt downstream targets. We show that OBD9 treatment inhibits colon cancer cell growth and both qPCR and Western blot data suggest that Wnt signaling downstream targets, such as TCF4, AXIN2 and cMyc, are all significantly suppressed by OBD9 via the inhibition of TNIK. Overall, our in vitro and in vivo data suggest that OBD9 potentially represents a novel therapeutic option for multiple cancers including but not limit to colon, lung and prostate cancer. Citation Format: Kun Zhou, Jae Eun Cheong, Catherine Alix-Panabières, Lijun Sun, Bruce R. Zetter. A novel orally available Wnt-pathway inhibitor for the treatment of metastatic cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 508.

Metabolites of intestinal microflora upregulate miR-192-5p to suppress proliferation of colon cancer cells via RhoA-ROCK-LIMK2 pathway.

The application of intestinal microflora is involved in various cancers; however, researches reporting the potential of metabolites of intestinal microflora (MIM) on biological activities of colon cancer (CC) cells are unavailable. This study was designed to testify the functions of MIM on CC cells and its mechanism. qRT-PCR/Western blot were applied to test the expression levels of miR-192-5p and BMPR2 in human colonic epithelial cells and CC cells (HCT116, SW480). The effects of MIM, mimics-miR-192-5p or inhibitors-miR-192-5p on mRNA and protein expressions of miR-192-5p and BMPR2 were verified by qRT-PCR and Western blot. MTT assay for CC cell viability, flow cytometry for CC cells apoptosis rate, and cell scratch and cell chamber served for the analysis of invasion and migration ability of CC cells. The relationship between miR-192-5p and BMPR2 was validated employing Luciferase reporter gene assay. Compared with human normal colonic epithelial cells, HCT116 and SW480 cells had lower expression of miR-192-5p and higher expression of BMPR2 (p < 0.01). MIM and mimics-miR-192-5p could enhance cell apoptosis and suppress the migration and proliferation of CC cells. MIM were also found to up-regulate miR-192-5p and down-regulate the expression levels of BMPR2 and p-LIMK2 (p < 0.01). Transfection of inhibitors-miR-192-5p reversed the inhibitory effect of MIM on CC cells. MIM could up-regulate miR-192-5p to inhibit CC cell growth via down-regulating BMPR2 and inhibiting the activity of RhoA-ROCK-LIMK2 pathway.

Matrine Reverses the Warburg Effect and Suppresses Colon Cancer Cell Growth via Negatively Regulating HIF-1α.

The Warburg effect is a peculiar feature of cancer’s metabolism, which is an attractive therapeutic target that could aim tumor cells while sparing normal tissue. Matrine is an alkaloid extracted from the herb root of a traditional Chinese medicine, Sophora flavescens Ait. Matrine has been reported to have selective cytotoxicity toward cancer cells but with elusive mechanisms. Here, we reported that matrine was able to reverse the Warburg effect (inhibiting glucose uptake and lactate production) and suppress the growth of human colon cancer cells in vitro and in vivo. Mechanistically, we revealed that matrine significantly decreased the messenger RNA (mRNA) and protein expression of HIF-1α, a critical transcription factor in reprogramming cancer metabolism toward the Warburg effect. As a result, the expression levels of GLUT1, HK2, and LDHA, the downstream targets of HIF-1α in regulating glucose metabolism, were dramatically inhibited by matrine. Moreover, this inhibitory effect of matrine was significantly attenuated when HIF-1α was knocked down or exogenous overexpressed in colon cancer cells. Together, our results revealed that matrine inhibits colon cancer cell growth via suppression of HIF-1α expression and its downstream regulation of Warburg effect. Matrine could be further developed as an antitumor agent targeting the HIF-1α-mediated Warburg effect for colon cancer treatment.

Curcuminoid WZ26, a TrxR1 inhibitor, effectively inhibits colon cancer cell growth and enhances cisplatin-induced cell death through the induction of ROS

Colon cancer is one of the leading causes of cancer-related deaths. Chemotherapy has improved survival in patients with colon cancer, but has a narrow therapeutic window due to its toxicity. Therefore, novel therapies for colon cancer are urgently needed. We previously developed a curcumin analog WZ26 as an anti-cancer agent in pre-clinical evaluation. In the present study, we further explored the mechanism and target of WZ26 in colon cancer cells. Our results show that WZ26 targets thioredoxin reductase 1 (TrxR1) and increases cellular reactive oxygen species (ROS) levels, which results in the activation of JNK signaling pathway in human colon cancer cells. Furthermore, we found that WZ26 significantly enhances cisplatin-induced cell growth inhibition in colon cancer cells. WZ26 combined with cisplatin markedly increases the accumulation of ROS, and thereby induces DNA damage and activation of JNK signaling pathway. Pretreatment with antioxidant N-acetyl-l-cysteine (NAC) significantly abrogates the combined treatment-induced ROS generation, DNA damage and cell death. In addition, the activation of JNK signaling pathway prompted by WZ26 and cisplatin was also reversed by NAC pretreatment. In vivo, WZ26 combined with cisplatin significantly inhibits tumor growth in a colon cancer xenograft model. Remarkably, WZ26 attenuates the body weight loss evoked by cisplatin treatment. This study discloses a previously unrecognized mechanism underlying the biological activity of WZ26, and reveals that WZ26 and cisplatin combinational treatment might potentially become a more effective regimen in colon cancer therapy.

TIPE1 impairs stemness maintenance in colorectal cancer through directly targeting β-catenin.

TIPE1 (tumor necrosis factor-α-induced protein 8-like 1) contributes to cell death in diverse cancers. However, the expression and biological functions of TIPE1 in colon cancer remain unclear. In the present study, we report that TIPE1 was downregulated in colon cancer tissues and positively correlates with prognosis of colon cancer patients. TIPE1 overexpression significantly inhibits colon cancer cell growth both in vitro and in vivo through impairing stemness, accompanied with downregulation of the stemness-related markers, ALDH, CD133, CD44 and SOX-9. Mechanically, TIPE1 directly targets β-catenin and promotes β-catenin degradation in a protease-dependent manner, and Wnt/β-catenin signaling plays a crucial role during TIPE1-mediated stemness inhibition in colon cancer. These findings reveal that TIPE1 exerts anti-tumor effects in colon cancer and suggest that TIPE1 would be a therapeutic target for cancers.

Histone deacetylase 6 is overexpressed and promotes tumor growth of colon cancer through regulation of the MAPK/ERK signal pathway.

Purpose: To investigate the expression of histone deacetylase 6 (HDAC6) in colon cancer and its role in colon cancer cell growth and migration.Materials and methods: We detected the expression of HDAC6 in a colon cancer tissue chip using immunochemical staining, and analyzed the difference in HDAC6 expression between cancer and adjacent noncancerous tissues. Then, we explored the relationship between HDAC6 expression and patients’ clinicopathological characteristics and prognoses. In adidition, the role of HDAC6 in colon cancer cell growth and migration, as well as its potential related signal pathway, through HDAC6 knockdown was explored.Results: The immunochemical score of HDAC6 expression was higher in cancer tissue than in the adjacent noncancerous tissue (4.54 vs 3.08, P<0.005); similarly, as well as the rate of high HDAC6 expression was higher in cancer tissue than in the adjacent noncancerous tissue (71.1% vs 40.9%, P<0.001). Patients showing high HDAC6 expression had a shorter overall survival time. Additionally, Cox regression analysis showed that high HDAC6 expression was an independent risk factor for poor prognosis. HDAC6 knockdown decreased cell viability, colony formation, and number of migrated colon cancer cells (HCT116 and HT29); the expression of p-MEK, p-ERK, and p-AKT was also decreased, but had no influence on MEK, ERK, and AKT expression.Conclusion: HDAC6 is highly expressed in colon cancer and associated with a poor prognosis. HDAC6 knockdown inhibits colon cancer cell growth and migration, partly through the MAPK/ERK pathway.

LncRNA-UCA1 modulates progression of colon cancer through regulating the miR-28-5p/HOXB3 axis.

Emerging evidence has shown that the long noncoding RNA urothelial carcinoma-associated 1 (UCA1) plays a tumor-promoting role in colorectal cancer, while miR-28-5p shows tumor-inhibitory activity in several tumor types. However, the mechanisms both of these in colon cancer progression are still unknown. In this work, the detailed roles and mechanisms of UCA1 and its target genes in colon cancer were studied. The results showed that UCA1 was upregulated in colon cancer tissues when compared with the adjacent nonhumorous tissues, as well as in the various colon cancer cell lines, but the expression of miR-28-5p showed an opposite trend. Furthermore, a high UCA1 level in colon cancer tissues is positively associated with the tumor size and advanced tumor stages. Functional assays revealed that both UCA1 knockdown and miR-28-5p overexpression could inhibit colon cancer cell growth and migration. Further mechanistic studies indicated that UCA1 knockdown played tumor suppressive roles in SW480 and HT116 cells through binding with miR-28-5p. We also, for the first time, identified HOXB3 as the target gene of miR-28-5p and that HOXB3 overexpression could mediate the functions of UCA1 in cell proliferation and migration of colon cancer cells. In conclusion, our data provided evidence for the regulatory network of UCA1/miR-28-5p/HOXB3 in colon cancer, suggesting that UCA1, miR-28-5p, and HOXB3 are the potential targets for colon cancer therapy.

Estrogen Receptor Beta (ERβ) Mediated-CyclinD1 Degradation via Autophagy Plays an Anti-Proliferation Role in Colon Cells.

Dysfunction of autophagic degradation machinery causes tumorigenesis, including colorectal cancer (CRC). Overexpression of CyclinD1 in CRC has been reported. Recent evidence also suggests that ERβ deficiency is related to the pathogenesis of CRC. Very little is known, however, about the detailed molecular mechanisms underlying the relationship among ERβ, autophagy, and CyclinD1 in CRC. Here, results showed that ERβ played an anti-proliferation role in HCT116 through impairing cell cycle but not apoptosis. Additionally, CyclinD1 accumulation was increased in response to chloroquine (CQ) or in MEF Atg7 knockout cells. Further, ERβ could inhibit the mammalian target of rapamycin (mTOR) or activate Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) to promote autophagy in HCT116. In summary, these results indicate that ERβ-mediated CyclinD1 degradation can inhibit colon cancer cell growth via autophagy.

Cordycepin induces Bax‑dependent apoptosis in colorectal cancer cells.

Cordycepin, or 3′-deoxyadenosine, is a derivative of the nucleoside adenosine. Initially extracted from the fungus Cordyceps militaris, cordycepin exhibits antitumor activity against certain cancer cell lines; however, the mechanism by which cordycepin counteracts colorectal cancer (CRC) remains poorly understood. The aim of the present study was to explore the underlying mechanisms of cordycepin against human CRC. To investigate the molecular mechanisms of cordycepin against colon cancer and in driving apoptosis, p53 and Bcl-2-like protein 4-null (Bax−/−) colon cancer HCT116 cell lines were used. Cell viability and growth were repressed in a dose-dependent manner in cells treated with cordycepin. Treatment with cordycepin resulted in increased apoptosis in HCT116 cells; however, flow cytometic analysis demonstrated that apoptosis was notably decreased in the Bax−/− HCT116 cell lines, but not in the p53−/− HCT116 cell lines. Furthermore, cordycepin exposure resulted in the translocation of Bax from the cytosol to the mitochondria and the subsequent release of cytochrome c from the mitochondria. Results from the present study demonstrated that cordycepin inhibited colon cancer cell growth in vitro and this appears to be through the endogenous Bax-dependent mitochondrial apoptosis pathway, which suggested a molecular mechanism for cordycepin against human colon cancer. These results indicated the possibility of cordycepin as a novel drug for the prevention of colon cancer.

Dihomo-\u03b3-linolenic acid inhibits xenograft tumor growth in mice bearing shRNA-transfected HCA-7 cells targeting delta-5-desaturase

BackgroundWe previously demonstrated that knockdown of delta-5-desaturase via siRNA transfection together with dihomo-γ-linolenic acid supplementation inhibited colon cancer cell growth and migration, by promoting the production of the anti-cancer byproduct 8-hydroxyoctanoic acid from Cyclooxygenase-2-catalyzed dihomo-γ-linolenic acid peroxidation. Here, we extend our study to investigate the effects of delta-5-desaturase-knockdown and the resulting intensified dihomo-γ-linolenic acid peroxidation in xenograft tumor mice model.MethodsFour-week old nude mice bearing the human colon cancer cell HCA-7/C29 vs. its delta-5-desaturase knockdown analog (via shRNA transfection) were subject to 4-week treatments of: vehicle control, dihomo-γ-linolenic acid supplementation, 5-Fluorouracil, and combination of dihomo-γ-linolenic acid and 5-Fluorouracil. Tumor growth was monitored during the treatment. At the endpoint, the mice were euthanized and the tumor tissues were collected for further mechanism analysis.ResultsDelta-5-desaturase knockdown (shRNA) together with dihomo-γ-linolenic acid supplementation increased 8-hydroxyoctanoic acid production to a threshold level in xenograft tumors, which consequently induced p53-dependent apoptosis and reduced tumors significantly. The promoted 8-hydroxyoctanoic acid formation was also found to suppress the tumors’ metastatic potential via regulating MMP-2 and E-cadherin expressions. In addition, our in vivo data showed that delta-5-desaturase knockdown along with dihomo-γ-linolenic acid supplementation resulted in anti-tumor effects comparable to those of 5-Fluorouracil.ConclusionsWe have demonstrated that our paradigm-shifting strategy of knocking down delta-5-desaturase and taking advantage of overexpressed Cyclooxygenase-2 in tumor cells can be used for colon cancer suppression. Our research outcome will lead us to develop a better and safer anti-cancer therapy for patients.

Both Bupivacaine and Levobupivacaine inhibit colon cancer cell growth but not melanoma cells in vitro

BackgroundRetrospective studies indicate that the use of regional anaesthesia causes a reduction in cancer recurrence after oncological surgery, which could be due to anaesthetic’s negating effect on immunosuppression related to the surgical stress response. Local anaesthetics may also exert direct suppressive effects on malignant cells, an area where further investigation is urgently needed.MethodsHuman colon cancer cells and human melanoma cells were cultured and then treated with 1 mM bupivacaine or levobupivacaine for up to 24 or 48 h. Their migratory ability was measured by scratch assay, proliferation determined with Ki67 immunofluorescence staining, and apoptosis accessed with annexin V and PI staining on flow cytometry. The effects of bupivacaine and levobupivacaine on cellular signaling and molecular response, specifically, on endoplasmic reticulum stress (ERS), were studied with immunostaining and western blot.ResultsIn colon cancer cells, treatment with bupivacaine and levobupivacaine significantly inhibited cell migration (**p < 0.01, ***p < 0.001; n = 4) and proliferation (**p < 0.01; n = 4), while increasing the expression of CHOP (***p < 0.001; n = 4) and decreased the expression of Grp78 (*p < 0.05; n = 4). These effects were not mirrored by melanoma cells, such that no significant increase in apoptosis was seen in either melanoma cell lines following treatment.ConclusionThese in vitro data suggested that both bupivacaine and levobupivacaine suppress colorectal adenocarcinoma cell proliferation and migration, which are concurrent with increased endoplasmic reticulum stress. Conversely, melanoma cells are more resilient to these two commonly used local anaesthetics. Further in vivo studies or clinical trials are needed.

MicroRNA 429 regulates the expression of CHMP5 in the inflammatory colitis and colorectal cancer cells.

MicroRNAs (miRNAs) play an important role in the pathogenesis of human diseases by regulating the expression of target genes in specific cells or tissues. In this study, we analyzed the association between the MIR429 and its target gene, charged multivesicular body protein 5 (CHMP5), in human colon cancer cells and in a DSS-induced colitis mouse model. A luciferase reporter system was used to confirm the effect of MIR429 on CHMP5 expression. Protein or mRNA expression of the target gene and associated molecules were measured by Western blot or quantitative RT-PCR (qRT-PCR), respectively. Flow cytometry was used to compare cell viability or cell cycle progression. CHMP5 mRNA and protein expression was directly down-regulated by MIR429. We found that MIR429 inhibited colon cancer cell growth and cell cycle progression, and CHMP5 was overexpressed in the DSS-induced colitis mouse model and human ulcerative colitis (UC) tissues. Our findings show that CHMP5 is a direct target of MIR429 in human colon cancer cell lines and suggest that CHMP5 up-regulation as a result of reduced MIR429 expression in DSS-induced mice colitis tissues and human UC tissues may restrict apoptosis and promote cell proliferation.

The Purified Extract from the Medicinal Plant Bacopa monnieri, Bacopaside II, Inhibits Growth of Colon Cancer Cells In Vitro by Inducing Cell Cycle Arrest and Apoptosis.

Aquaporin-1 (AQP1), a transmembrane pore-forming molecule, facilitates the rapid movement of water and small solutes across cell membranes. We have previously shown that bacopaside II, an extract from the medicinal herb Bacopa monnieri, blocks the AQP1 water channel and impairs migration of cells that express AQP1. The aim of this study was to further elucidate the anti-tumour potential of bacopaside II in colon cancer cells. Expression of AQP1 in HT-29, SW480, SW620 and HCT116 was determined by quantitative PCR and western immunoblot. Cells were treated with bacopaside II, and morphology, growth, autophagy, cell cycle and apoptosis assessed by time-lapse microscopy, crystal violet, acridine orange, propidium iodide (PI) and annexin V/PI staining respectively. AQP1 expression was significantly higher in HT-29 than SW480, SW620 and HCT116. Bacopaside II significantly reduced growth at ≥20 µM for HT-29 and ≥15 µM for SW480, SW620 and HCT116. Inhibition of HT-29 at 20 µM was primarily mediated by G0/G1 cell cycle arrest, and at 30 µM by G2/M arrest and apoptosis. Inhibition of SW480, SW620 and HCT116 at ≥15 µM was mediated by G2/M arrest and apoptosis. These results are the first to show that bacopaside II inhibits colon cancer cell growth by inducing cell cycle arrest and apoptosis.

Abstract 3494: Downregulation of SREBPs inhibited colon cancer cell growth and tumor initiation by alteration of metabolism

Abstract Sterol regulatory element binding proteins (SREBPs) belong to a family of transcription factors that regulate the expression of genes required for the synthesis of fatty acids and cholesterol. There are three SREBP isoforms, SREBP1a, SREBP1c and SREBP2, have been identified in mammalian cells. SREBP1a and SREBP1c are derived from a single gene through the use of alternative transcription start sites. Increasing evidence suggests that increased lipogenesis is needed for cancer cells to accommodate high rates of proliferation. Activation of SREBP1 and enhanced expression of its target genes have been observed in different types of human cancers. However, the functional importance of SREBPs in colon cancer remains to be fully elucidated. In this study, we investigated the role of SREBP-mediated lipid biosynthesis in promoting tumor growth and initiation by regulating cellular metabolism in colon cancer. Knockdown of SREBP-1 or SREBP-2 decreased the expression of target genes required for lipid biosynthesis and levels of fatty acids and triacylglycerides in colon cancer cells. Bioenergetic analysis using Seahorse XF96 Extracelluar Flux Analyzer revealed that silencing SREBP-1 or SREBP-2 expression reduced the mitochondrial respiration, glycolysis, as well as fatty acid oxidation suggesting an inhibition of cellular metabolism. As a result, the rate of cell proliferation and the ability of cancer cells to form tumor spheroids in suspension were decreased. Similar results were obtained in colon cancer cells in which SCAP, an upstream protease responsible for activating SREBP-1 and SREBP-2, was silenced. Mechanistically, downregulation of SREBPs inhibited the activation of AMPK and Akt signaling. Furthermore, knockdown of SREBP-1 or SREBP-2 inhibited xenograft tumor growth in vivo and significantly decreased the expression of genes associated cancer stem cells. Taken together, our findings establish the molecular basis of SREBPs-dependent metabolic regulation and provide a rationale for targeting fatty acid synthesis as a promising approach in colon cancer treatment. Citation Format: Yang-an Wen, Xiaopeng Xiong, Yekaterina Zaytseva1, Tianyan Gao. Downregulation of SREBPs inhibited colon cancer cell growth and tumor initiation by alteration of metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3494.

Abstract 4514: ASR458, a small molecule, targets Notch1 signaling in advanced colorectal cancer

Abstract Cancer-related mortality rates are amongst the highest in colorectal cancer (CRC). Previously we have reported that Withaferin A (WA), a natural compound, induces apoptosis and inhibits AKT mediated pro-survival signaling resulting in growth inhibition both in vitro and in vivo models of colon cancer. Owing to the encouraging results with WA, we have developed a series of acetylated pro-drug conjugates of WA and our initial structure-activity relationship (SAR) studies led to the identification of a novel small molecule ASR-458 as the lead compound. Interestingly, ASR-458 has demonstrated better efficacy by inhibiting colon cancer cell growth at nano-molar concentration, even in AKT overexpressing (AKT/HCT) HCT-116 cells (IC50: 750nm). Induction of apoptosis was seen in both control vector and AKT overexpressed colon cancer cell lines. Induction of caspase mediated signaling confirmed the pro-apoptotic function of ASR-458 in colon cancer cells. Inhibition of pAKT (ser473) resulted in downregulation of Notch1 downstream signaling pathways (Presenilin 1, NFkB activation and BCl-2 expression) in AKT/HCT colon cancer cells. Epithelial to mesenchymal transition (EMT) is a major hallmark of metastatic colon cancer. ASR-458 also downregulated the expression of EMT markers snail as well as β-Catenin and significantly impaired invasive as well as the migratory efficiency of AKT/HCT cells. Our ongoing xenograft studies will validate the in vivo efficacy of ASR-458 (1mg/kg/BW by IP weekly 2 times for six weeks) will confirm the chemotherapeutic effect of the compound against colon cancer cells. Overall these results emphasize ASR-458 to be a promising small molecule as compared to WA. ASR-458 targets both AKT and Notch1 signaling pathways which remains an important target for the development of effective treatment of metastatic stage of CRC. Citation Format: Balaji Chandrasekaran, Deeksha Pal, Ashish Tyagi, Srinivasa R Ramisetti, Arun K Sharma, Murali Ankem, chendil Damodaran. ASR458, a small molecule, targets Notch1 signaling in advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4514.