HT-29 Colon Cancer Cells Research Articles

Inhibitors of Gq signalling down-regulate β-catenin expression & function in human colon cancer cells.

Background & objectives:β-catenin signalling plays a key role in maintaining normal cellular physiology, and therefore, its deregulation can lead to many human diseases including cancers. Previously, we have shown that the activation of Gq signalling positively regulates β-catenin by inhibiting glycogen synthase kinase-3 beta and increasing the stability of β-catenin protein, however, these results were mainly based on overexpression experiments in either Xenopus oocytes or HEK293T cells. The present study was undertaken to evaluate the modulation of Gq signalling in human colon cancer cells.Methods:Gq signalling in SW480 and HT-29 colon cancer cells was specifically blocked to investigate the interaction between β-catenin and the Gq signalling pathways. GP antagonist-2A (a commercially available peptide) and a minigene expression construct encoding a peptide corresponding to the C-terminal 11 amino acids of Gαq were used to block Gq signalling. β-catenin expression and function were examined by western blotting, immunofluorescence microscopy, and quantitative real-time PCR experiments.Results:Transfection of cells with either of the blockers significantly decreased both β-catenin protein levels and β-catenin-mediated transcriptional activities. In addition, the migration of SW480 cells was reduced in the presence of the Gq blockers.Interpretation & conclusions:The results of this study further support the positive role of Gq signalling in regulating β-catenin expression and function and may provide a new means of preventing β-catenin-mediated carcinogenesis by blocking heterotrimeric G proteins.

Polyphyllin I inhibit cell viability, migration and invasion of human colon cancer cells by regulating MiR-26a/TGF-β signaling pathway

miR-26a is reported to be involved in both tumor suppressor or oncogenic action in various types of cancer progression. Colon cancer is the fourth leading lethal form of cancer and cause large number of deaths worldwide. Despite its various health beneficial effects against abnormalities, Polyphyllin I (PPI), has lack of scientific evidence on its role in colon cancer therapeutic efficacy were entice our attention. This study is designed to identify the role of miR-26a in the PPI mediated treatment for colon cancer. Further, Gene Ontology (GO) data on miR-26a biological process of its association with Transforming Growth Factor (TGF-β) signaling also hypothesized. In the present study, the protective role of PPI in cell migration and invasion of human colon cancer SW-948 and HT-29 cells was determined assays method. Further, reverse transcription-quantitative Polymerase Chain Reaction (qRT-PCR) analysis was carried out to determine the expression pattern of miR-26a and TGF-β, subsequently western blot analysis was performed to determine the TGF-β expression pattern. This experimental analysis validates the therapeutic efficiency of PPI that whether the anticancer potential was mediated by Transforming Growth Factor-β (TGF-β) cascade signaling and microRNA (miR) 26a. Earlier, Cell viability assay was performed to identify the Minimum inhibitory concentration M (IC50) of PPI on colon cancer cells. Our results suggest that cell proliferation, migration and invasion of cancer cells (SW-948 and HT-29) was significantly suppressed by PPI treatment, further the effect was observed in a dose-dependent manner. Moreover, following PPI treatment, miR-26a was up-regulated were further induce the tumor suppressor property also. This process consequently alters the TGF-β signaling cascade process that reportedly complicated, were associate with cell proliferation and differentiation. Findings of this study elucidated that, the over expression of miR-26a subsequently downstream the TGF-β signaling pathway, consequence of this effect was found to be PPI treated colon cancer cells. This might be the possible mechanism of action of PPI by which appears to be produce effective therapeutic potential in colon cancer therapy.

In silico and in vitro design of cordycepin encapsulation in liposomes for colon cancer treatment.

Cordycepin or 3′-deoxyadenosine is an interesting anti-cancer drug candidate that is found in abundance in the fungus Cordyceps militaris. It inhibits cellular growth of many cancers including lung carcinoma, melanoma, bladder cancer, and colon cancer by inducing apoptosis, anti-proliferation, anti-metastasis and by arresting the cell cycle. Cordycepin has, however, poor stability and low solubility in water, resulting in loss of its bioactivity. Liposomes can be used to overcome these obstacles. Our aim is to improve cordycepin's anti-colon cancer activity by liposome encapsulation. Cordycepin-encapsulated liposomes were designed and fabricated based on a combination of theoretical and experimental studies. Molecular dynamics (MD) simulations and free energy calculations suggest that phosphatidylcholine (PC) lipid environment is favorable for cordycepin adsorption. Cordycepin passively permeates into PC lipid bilayers without membrane damage and strongly binds to the lipids' polar groups by flipping its deoxyribose sugar toward the bilayer center. Our fabricated liposomes containing 10 : 1 molar ratio of egg yolk PC : cholesterol showed encapsulation efficiency (%EE) of 99% using microfluidic hydrodynamic focusing (MHF) methods. In our in vitro study using the HT-29 colon cancer cell line, cordycepin was able to inhibit growth by induction of apoptosis. Cell viability was significantly decreased below 50% at 125 μg mL−1 dosage after 48 h treatment with non-encapsulated and encapsulated cordycepin. Importantly, encapsulation provided (1) a 2-fold improvement in the inhibition of cancer cell growth at 125 μg mL−1 dosage and (2) 4-fold increase in release time. These in silico and in vitro studies indicate that cordycepin-encapsulated liposomes could be a potent drug candidate for colon cancer therapy.

Chemopreventive effects of vitamin D3 and its analogue, paricalcitol, in combination with 5-fluorouracil against colorectal cancer: The role of calcium signalling molecules

BackgroundAlthough vitamin D (VD) is chemoprotective and enhances 5-fluorouracil (5-FU) cytotoxicity against colorectal cancer (CRC), little is known about its potential calcium (Ca2+)-mediated anti-tumorigenic actions. Therefore, this study compared between VD and its non-calcaemic analogue, Paricalcitol (Pcal), ± 5-FU in relation to chemoprevention and Ca2+-mediated apoptosis in vivo and in vitro. MethodsSeventy male mice were distributed to: negative controls, positive controls (PC), VD, Pcal, 5-FU, VD + 5-FU and Pcal+5-FU groups. All groups, except negative, received two consecutive azoxymethane (AOM)-injections (10 mg/Kg/week) for CRC induction. VD3 (1000 IU/kg; three times/week) and Pcal (1.25 μg/kg; three times/week) injections started week-16 post-AOM and for 10 weeks. Three successive 5-FU cycles began at week-21 (50 mg/Kg/week). Similar protocols with VD3, Pcal and/or 5-FU were applied in the HT29 colon cancer cells. ResultsThe PC group had abundant malignant tumours, markedly elevated proliferation markers (survivin/CCND1) and declines in cyclin-dependent kinase-inhibitor-1A, pro-apoptotic molecules (p53/BAX/cytochrome_C/caspase-3), tissue Ca2+ concentrations and Ca2+-dependent proteins (CaSR/CAM/CAMKIIA). All monotherapies equally reduced tumour numbers and proliferation markers whilst promoting the anti-tumorigenic molecules. VD and/or 5-FU, but not Pcal monotherapy, enhanced Ca2+ levels and Ca2+-related molecules (CaSR/CAM/CAMKIIA/BAX/cytochrome_C) in vivo and in vitro. However, VD + 5-FU co-therapy showed the lowest tumour numbers, the highest cell numbers in sub-G1 phase of cell cycle, alongside the most effective modulations of oncogenes, tumour suppressors and Ca2+-related molecules at the gene and protein levels in vivo and in vitro. ConclusionsVD3 was superior than Paricalcitol in potentiating 5-FU cytotoxicity, possibly by upregulating several Ca2+-related molecules involved in tumour suppression.

Mechanical characterization of bio composite films as a novel drug carrier platform for sustained release of 5-fluorouracil for colon cancer: Methodological investigation

In this study, we employed Pectin (PC) as a matrix that is hybridized with three different nucleobase (NB) units (cytosine, thymine, uracil) to generate pectin–nucleobase(PC-NB) biocomposite films stabilized through bio-multiple hydrogen bonds (BMHBs) as drug carrier for anticancer 5-Fluorouracil (5-FU). Prepared biocomposite films were characterized by Fourier Transform Infra-red Spectroscopy (FTIR), X-ray Diffraction (XRD), Thermogravimmetry Analysis (TGA) and Scanning Electron Microscope (SEM). Mechanical and sorption properties were also evaluated. In vitro drug release performed in both acidic pH 1.2 (stomach pH) and alkaline pH 7.4 (intestinal pH) showed that incorporation of nucleobases into pectin significantly restricted release rate of 5-FU particularly under acidic condition (pH 1.2). Hemolysis assays demonstrated that PC-NB-5-FU biocomposite film drug carriers were hemocompatible. Confocal microscope analysis indicates facilitated cellular uptake of PC-NB-5-FU film in HT-29 colon cancer cell line, which in turn result in a higher potential of apoptosis. Confocal imaging of fluorescent live/dead cell indicators and MTT assay outcomes, both demonstrated significant decreases in cellular viability of PC-NB-5-FU biocomposite films. Collectively, our findings indicate that this PC-NB-5-FU biocomposite films can be conferred as a proficient formulation for targeted delivery of colon cancer drugs.

PLGA-Based Nano-Encapsulation of Trachyspermum Ammi Seed Essential Oil (TSEO-PNP) as a Safe, Natural, Efficient, Anticancer Compound in Human HT-29 Colon Cancer Cell Line

Colorectal cancer is a lethal and commonly diagnosed cancer worldwide. To halt its burden more efficient targeted strategies are needed. Trachyspermum ammi seed essential oil (TSEO) contains several anticancer phytochemicals that maybe more effective via PLGA-based nano-encapsulation. TSEO-PNP nanoparticles were synthesized utilizing evaporation and ultra-sonication-based emulsification methods. Their size, morphology, and stability were defined by DLS, SEM, and surface zeta-potential data, respectively. The TSEO-PNP antioxidant apoptotic, cytotoxic, and antiangiogenic impacts on both cell lines (HT-29 and HUVEC) were studied by FRAP/ABTS, Q-PCR, MTT, and CAM assays, respectively. Moreover, further confirmatory measurements such as AO/EB fluorescent staining and flow cytometry analysis were performed to verify apoptosis. Stable (-32.42 mV) 206.21-nm TSEO-PNP induced apoptosis in the HT-29 cells. Apoptosis was confirmed by significant overexpression of apoptotic genes (Cas-9 and BAX), down-regulation of the anti-apoptotic (BCL-2) gene, fluorescent AO/EB staining, and flow cytometry data following increased TSEO-PNP treatment doses. TSEO-PNP exhibited a meaningful dose- and time-dependent cancer-specific cytotoxic impact on HT-29 cells. The TSEO-PNP has three main anticancer activities on HT-29 colon cancer cells including oxidant reduction, apoptosis induction, and angiogenesis suppression.

Asperginols A and B, Diterpene Pyrones, from an Aspergillus sp. and the Structure Revision of Previously Reported Analogues.

Two new diterpene pyrones, asperginols A (1) and B (2), and four known analogues (3-6) were isolated from the endophytic fungus Aspergillus sp. HAB10R12. The structures and absolute configurations of these compounds were elucidated based on the analysis of their NMR, MS, and X-ray diffraction data. The revision of the absolute configurations at C-10, C-11, and C-14 of the known diterpene pyrones (3-6) and the determination of the configuration at the polyene side chain for compounds (4-6) were made using chemical methods and vibrational circular dichroism analysis. This group of diterpene pyrone compounds showed unique structural features including a 7/6/6 tricyclic diterpene moiety with an unusual trans-syn-trans stereochemical arrangement. Compound 6 showed moderate activity against the HT-29 colon cancer cell line.

Oncolytic effects of the recombinant Newcastle disease virus, rAF-IL12, against colon cancer cells in vitro and in tumor-challenged NCr-Foxn1nu nude mice.

Colon cancer remains one of the main cancers causing death in men and women worldwide as certain colon cancer subtypes are resistant to conventional treatments and the development of new cancer therapies remains elusive. Alternative modalities such as the use of viral-based therapeutic cancer vaccine is still limited, with only the herpes simplex virus (HSV) expressing granulocyte-macrophage colony- stimulating factor (GM-CSF) or talimogene laherparepvec (T-Vec) being approved in the USA and Europe so far. Therefore, it is imperative to continue the search for a new treatment modality. This current study evaluates a combinatorial therapy between the oncolytic Newcastle disease virus (NDV) and interleukin-12 (IL-12) cytokine as a potential therapeutic vaccine to the current anti-cancer drugs. Several in vitro analyses such as MTT assay, Annexin V/FITC flow cytometry, and cell cycle assay were performed to evaluate the cytotoxicity effect of recombinant NDV, rAF-IL12. Meanwhile, serum cytokine, serum biochemical, histopathology of organs and TUNEL assay were carried out to assess the anti-tumoral effects of rAF-IL12 in HT29 tumor-challenged nude mice. The apoptosis mechanism underlying the effect of rAF-IL12 treatment was also investigated using NanoString Gene expression analysis. The recombinant NDV, rAF-IL12 replicated in HT29 colon cancer cells as did its parental virus, AF2240-i. The rAF-IL12 treatment had slightly better cytotoxicity effects towards HT29 cancer cells when compared to the AF2240-i as revealed by the MTT, Annexin V FITC and cell cycle assay. Meanwhile, the 28-day treatment with rAF-IL12 had significantly (p < 0.05) perturbed the growth and progression of HT29 tumor in NCr-Foxn1nu nude mice when compared to the untreated and parental wild-type NDV strain AF2240-i. The rAF-IL12 also modulated the immune system in nude mice by significantly (p < 0.05) increased the level of IL-2, IL-12, and IFN-γ cytokines. Treatment with rAF-IL12 had also significantly (p < 0.05) increased the expression level of apoptosis-related genes such as Fas, caspase-8, BID, BAX, Smad3 and granzyme B in vitro and in vivo. Besides, rAF-IL12 intra-tumoral delivery was considered safe and was not hazardous to the host as evidenced in pathophysiology of the normal tissues and organs of the mice as well as from the serum biochemistry profile of liver and kidney. Therefore, this study proves that rAF-IL12 had better cytotoxicity effects than its parental AF2240-i and could potentially be an ideal treatment for colon cancer in the near future.

Synthesis of 3-Trifluoromethyl-5,6-dihydro-[1,2,4]triazolo Pyrazine Derivatives and Their Anti-Cancer Studies

The synthesis of a wide variety of 3-trifluoromethyl-5,6-dihydro-[1,2,4]triazolo pyrazine derivatives, by the treatment of 3-trifluoromethyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-α]pyrazine hydrochloride with an array of isocyanates in the presence of triethylamine, is reported. All the target compounds were synthesized in excellent yields under mild reaction conditions. The target molecules were effectively screened for their anti-cancer properties and the results are promising. The resultant compounds were assessed for their antiproliferative action against two human colon cancer cell lines (HCT-116 and HT-29 colon cancer cell lines). The IC50 range was estimated at 6.587 to 11.10 µM showing that compound RB7 had remarkable anticancer movement on HT-29. Additionally, it was discovered that RB7 incited the mitochondrial apoptotic pathway by up-regulating Bax and down-regulating Bcl2, eventually leading to the activation of Caspase 3 in HT-29 cells and initiation of cell death via the mitochondrial apoptotic pathway.

Multi-Species Probiotic Modulates Cytokine Production and the Interplay between Immune and Colon Cancer Cells

The current study aimed to investigate the effect of a multi-species probiotic (MSP) on cytokine production by human peripheral blood mononuclear cells (PBMCs) and their immune dialogue with HT-29 colon cancer cells. PBMCs were incubated with MSP and their effect on cell proliferation and TNFα, IL-1β, IL-2, IL-6, IFNγ, IL-10, and IL-1ra production was evaluated. The impact of MSP on the cytokine production by PBMC stimulated by HT-29 cells was detected. Not-stimulated PBMC incubated with MSP showed increased production of TNFα, IL-1β, IL-6, and IL-10, but no change in IL-6, IFNγ, and IL-1ra. The stimulatory effect of MSP on lipopolysaccharide (LPS)-promoted PBMC was less pronounced for TNFα, IL-1β, and IFNγ, and the IL-6 production was decreased; phorbol 12-myristate 13- acetate (PMA)-induced IL-2 and IFNγ secretion was inhibited. The addition of MSP to co-cultures of PBMC and HT-29 cancer cells caused a remarkable increase in TNFα and IL-1β secretion, with no change in remaining cytokines. The multi-species probiotics modulated cytokine production by PBMC and affected the cross-talk between PBMC and HT-29 cancer cells. We conclude that probiotics may serve as supplements to the therapeutic strategies applied for the treatment of chronic inflammatory and malignant diseases, especially colorectal cancers.

Cytotoxicity of metformin against HT29 colon cancer cells contributes to mitochondrial Sirt3 upregulation.

Cancer and diabetes, the two mitochondria-related diseases, have recently been linked to silent mating-type information regulation 2 homolog 3 (SIRT3) activity irregularities. In this study, the effect of metformin, an antidiabetic with anticancer properties, has been evaluated on mitochondrial functionality markers, cell death pathways, and SIRT3 enzyme activity in the colon cancer cell line,HT-29, and human embryonic kidney cells (HEK 293). HT-29 cells were treated with metformin (5, 10, 20, 40, and 80 µM) for 24, 48, and 72 h for measuring the IC50 concentration. Reactive oxygen species (ROS) production, apoptosis, mitochondrial membrane potential, SIRT3 activity, and expression were evaluated against the colon cancer cell line, HT-29. Results indicated a higher ROS production at 6 than 12 h with metformin treatment. Metformin modified the mitochondrial membrane potential, resulting in cell death induction. Results from SIRT3 activity and expression showed that metformin increased its activity and expression in cancer cells. In conclusion, metformin in HT-29 cells disturbed the mitochondrial activity via increased ROS levels and SIRT3 activity, and these rapid modifications may play a key role in its cytotoxic property.

Overexpression of Pyruvate Carboxylase Is Correlated With Colorectal Cancer Progression and Supports Growth of Invasive Colon Cancer HT-29 Cell Line.

Pyruvate carboxylase (PC) is a major anaplerotic enzyme for generating oxaloacetate for the TCA cycle and also a key enzyme in gluconeogenesis, de novo fatty acid and amino acid synthesis in normal cells. Recent studies have identified PC overexpression in different cancers, such as breast and lung. However, the involvement of PC in colorectal cancer (CRC) is unclear. Our purpose was to investigate the PC expression levels and its correlations with potentially relevant clinical-pathological parameters in CRC. PC expression levels in tissues from 60 Thai CRC patients were investigated by immunohistochemistry while a clonogenic assay was performed for determining cell growth of HT-29 cells with PC knockdown. Our results showed for the first time that high PC expression levels were significantly correlated with late stage of the cancer, perineural invasion and lymph node metastasis. The overexpression of PC was also significantly associated with poor overall and disease-free survival times of CRC patients. In addition, suppression of cancer cell growth was found in PC-deficient cell lines using CRISPR-Cas9. The overexpression levels of PC were correlated with CRC progression and survival times. Therefore, PC might serve as a potential clinical prognostic marker for colorectal cancer.

A ROS-Responsive Self-Assembly Driven by Multiple Intermolecular Interaction Enhances Tumor-Targeted Chemotherapy.

To develop a drug carrier sensitive to reactive oxygen species (ROS), a nanocomplex (NCs) based on sulfuric hyaluronic acid (sHA)-anthocyanin (ATC) was developed. Doxorubicin (DOX) is loaded into the sHA-ATC NCs (AD@sHA) through intermolecular π-π stacking and hydrophobic interactions. AD@sHA can be prepared in aqueous phase by simple mixing method. In AD@sHA, DOX content and load efficiency are high. Compared with D@sHA (without ATC), the ROS-ATC co-mediated responsive degradation and drug release of AD@sHA was confirmed. In addition, AD@sHA also improved apoptosis of CD44+ colon cancer HT29cells. HT29 tumor-bearing mice model was used to confirm the role of AD@SHA in targeted tumor therapy. The results showed that AD@SHA could optimize the biodistribution of DOX. These data, from tumor volume and TUNEL analysis, observed the delay of tumor growth and apoptosis of cancer cells. Even more exciting is that AD@sHA significantly reduces the myelosuppression of DOX. This study means that AD@sHA has a better effect on chemotherapy for CD44-positive tumors.

Microwave-assisted extraction of phenolic compounds with antioxidant and anti-proliferative activities from supercritical CO2 pre-extracted mango peel as valorization strategy

This work includes the second/sequential stage of a green-based valorization strategy of mango peel. An exhausted biomass from a pilot-scale CO2 supercritical extraction process was reused for obtaining phenolic-rich extracts with high antioxidant and anti-proliferative activity, employing microwave-assisted extraction. The effects of microwave power (400–800 W), liquid-to-solid ratio (10–50 mL/g) and extraction time (60–120 s) on process yield, phenolic content, and antioxidant capacity were investigated using a Box-Behnken design. A solution consisting of 60% aqueous ethanol was used as extraction solvent. The results showed that microwave power and liquid-to-solid ratio were the most influential factors on the responses variables. The highest total phenolic content (52.08 mg gallic acid eq./g d. w.) and antioxidant activities (2.75 mmol trolox eq./g extract, and of 6.47 μg/mL expressed in DPPH, EC50) were obtained at 800 W, 50 g/mL, and 90 s. Mango peel extract recovered at optimal conditions provided high anti-proliferative activity against HT-29 colon cancer cells line, after 24 h treatment (IC50 = 22.98 μg/mL). Gallic acid derivatives, such as galloyl-esters, xanthones like mangiferin, flavonoids, including quercetin and quercetin glycosides were tentatively identified by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Most probably, the compounds responsible for the outstanding anti-proliferative activity.

Phytochemical screening, antioxidant, anti-diabetic and cytotoxic activity of leaves of Pandanus canaranus Warb

Pandanus, a traditional medicinal plant is used for the ailment of several diseases in India. The extract prepared from leaves was tested for its secondary metabolites and also to phenolics, alkaloids, saponins and flavonoids. Subsequently, the antioxidant activity was observed using DPPH, reducing power, FRAP, metal chelating activity and total antioxidant assays. It is evident that more secondary metabolites were present in ethyl acetate and methanol solvents enriched in free radicals scavenging ability compared with other solvents. Further, the extracts were tested and observed for potential presence of anti-diabetic activity with strong inhibition of α-amylase and α-glucosidase which suggests that the absorption and digestion of carbohydrates would possibly reduce the blood glucose levels and hence help management of type-2 diabetes. Though the ethyl acetate and methanol extract exhibited cytotoxicity and apoptosis activity on human HT-29 colon cancer cell line via MTT, AO/EtBr and DAPI staining assays that inhibits cancer cell line which in turn prevents cancer.

Comparison of anticarcinogenic properties of Viburnum opulus and its active compound p-coumaric acid on human colorectal carcinoma.

Resistance to therapeutic agents and the highly toxic side effects of synthetic drugs has spurred new research in the treatment of colon cancer, which has high morbidity and mortality ratios. This study aims to clarify the molecular mechanisms of the anticarcinogenic properties of methanol extract of Viburnum opulus L. (EVO)and its main active compound, trans-p -coumaric acid ( p -CA), on human colon cancer cells (DLD-1, HT-29, SW-620, Caco-2) and healthy colon epithelial cells (CCD-18Co). The effects of EVO on controlled cell death (apoptosis) and the cell division cycle were determined by flow cytometry. Alteration in mRNA and protein expressions of switch genes in colorectal carcinoma (APC, MLH1, TP53, SMAD4, KRAS, and BRAF) were determined by qRT-PCR and Western blot, respectively. Our results show that EVO possesses a strong reducing capacity and free-radical scavenging activity. HPLC analyses prove that p -CAis the main compound of EVO. EVO and p -CA inhibit the proliferation of human colon cancer cells DLD-1 and HT-29 in a dose-dependent manner. EVO increases apoptosis of DLD-1 cells and halts the cell cycle in the G2 stage in HT-29 cells. mRNA and protein expressions of p53 and SMAD-4 are upregulated, while BRAFs are downregulated. The results were directly proportional to p -CA. EVO and p -CA up- and downregulate switch genes and protein expressions of DLD-1 cells, which alter the expression of 186 other genes. This is the first study of pharmacological exploration of V.opulus in human colon cancer. Its antiproliferative effects may be due to the presence of p -CA.

MicroRNA-340-5p inhibits colon cancer cell migration via targeting of RhoA

Colon cancer is the third most common cancer and a significant cause of cancer-related deaths worldwide. Metastasis is the most insidious aspect of cancer progression. Convincing data suggest that microRNAs (miRs) play a key function in colon cancer biology. We examined the role of miR-340-5p in regulating RhoA expression as well as cell migration and invasion in colon cancer cells. Levels of miR-340-5p and RhoA mRNA varied inversely in serum-free and serum-grown HT-29 and AZ-97 colon cancer cells. It was found transfection with miR-340-5p not only decreased expression of RhoA mRNA and protein levels in HT-29 cells but also reduced colon cancer cell migration and invasion. Bioinformatics analysis predicted one putative binding sites at the 3′-UTR of RhoA mRNA. Targeting this binding site with a specific blocker reversed mimic miR-340-5p-induced inhibition of RhoA activation and colon cancer cell migration and invasion. These novel results suggest that miR-340-5p is an important regulator of colon cancer cell motility via targeting of RhoA and further experiments are warranted to evaluate the role of miR-340-5p in colon cancer metastasis.

Discovery of novel artemisinin-sulfonamide hybrids as potential carbonic anhydrase IX inhibitors with improved antiproliferative activities

A series of artemisinin-sulfonamide hybrids (1–16) have been designed and synthesized by using molecular hybridization approach and investigated for the inhibitory activity of four human (h) carbonic anhydrases (CAs, EC 4.2.1.1), hCA I, II, IX and XII. The results indicated most of the target compounds showed better CA IX and CA XII inhibitory activity than the starting segment sulfanilamide. Among all the compounds, compound 3 (IC50: 5 nM) showed the best CA IX inhibitory efficacy. The p-aminobenzenesulfonamide derivatives showed significant antiproliferative activities against MDA-MB-231 breast cancer cell line and HT-29 colon cancer cell line under hypoxic conditions where CA IX and CA XII are overexpressed and most of them showed no apparent cytotoxic effects toward MCF-10A normal mammary epithelial cell. Among these derivatives, compound 3 displayed the most potent antiproliferative activities (IC50: 0.65 μM) against HT-29 cell line under hypoxia and low cytotoxicity (IC50: 78.0 μM) toward normal cell line. Meanwhile, compound 3 was found to efficiently decrease the hypoxia-induced extracellular acidification in both cancer cells. Molecular docking studies of compounds 3, 4, 5 and 9 revealed the proper interactions between the hybrid molecules and the active site of CA IX. All the results proved the effectiveness of the hybridization approach to develop novel artemisinin-sulfonamide compounds targeting CA IX for cancer treatment.

Effect of the Cellular Extract of Co-cultured Lactobacillus Casei on BAX and Human β-Defensin 2 Genes Expression in HT29 Cells

Aims: Defensins are cysteine-rich antimicrobial cationic peptides and BAX is a proapoptotic gene that can cause cell death. This study aimed to investigate the effect of cellular extract of co-cultured Lactobacillus casei on the expression of BAX and human β-defensin 2 (hBD-2) genes in HT29 cells. Methods &amp; Materials: This experimental study was conducted in the Research Center for Pharmaceutical Nanotechnology of Tabriz University of Medical Sciences in 2017. The HT29 cell line was obtained from the Pasteur Institute of Iran, and cells were assessed using Microculture Tetrazolium Test (MTT) after culturing. DNA was extracted from the treated cells, and then the DNA ladder assay was carried out. After preparing cDNA, the expression levels of BAX and hBD-2 genes in the HT29 cell line were measured using a real-time Polymerase Chain Reaction (PCR) method. Findings: The results of the MTT assay indicated that Lactobacillus casei inhibited the proliferation of HT29 cells and induced apoptosis in these cells. Results of DAPI staining and DNA ladder assay obtained from treating HT29 cells by Lactobacillus casei showed qualitative changes in cell apoptosis. Moreover, realtime PCR results indicated that Lactobacillus casei bacteria significantly increased the expression of the hBD-2 gene in HT29 colon cancer cells within 12-24 hours (P= 0.023), while BAX gene expression showed no significant change in the first 24 hours (P= 0.37). Conclusion: The extract of Lactobacillus casei can be used to stimulate cancer cells to produce β-defensins, inhibit pathogens, prevent the stimulation of cellular signaling, and fight antibiotic-resistant bacteria.

The Study of Expression of Casp3/Bax Genes in HT29 Colon Cancer Cell Line with Lactobacillus rhamnosus Co-Culturing

The Study of Expression of Casp3/Bax Genes in HT29 Colon Cancer Cell Line with Lactobacillus rhamnosus Co-Culturing