Human Colorectal Carcinoma HCT116 Cells Research Articles

A nanosecond pulsed electric field (nsPEF) can affect membrane permeabilization and cellular viability in a 3D spheroids tumor model

Three-dimensional (3D) cellular models represent more realistically the complexity of in vivo tumors compared to 2D cultures. While 3D models were largely used in classical electroporation, the effects of nanosecond pulsed electric field (nsPEF) have been poorly investigated. In this study, we evaluated the biological effects induced by nsPEF on spheroid tumor model derived from the HCT-116 human colorectal carcinoma cell line. By varying the number of pulses (from 1 to 500) and the polarity (unipolar and bipolar), the response of nsPEF exposure (10 ns duration, 50 kV/cm) was assessed either immediately after the application of the pulses or over a period lasting up to 6 days. Membrane permeabilization and cellular death occurred following the application of at least 100 pulses. The extent of the response increased with the number of pulses, with a significant decrease of viability, 24 h post-exposure, when 250 and 500 pulses were applied. The effects were highly reduced when an equivalent number of bipolar pulses were delivered. This reduction was eliminated when a 100 ns interphase interval was introduced into the bipolar pulses. Altogether, our results show that nsPEF effects, previously observed at the single cell level, also occur in more realistic 3D tumor spheroids models.

Potential anticarcinogenic effect of goat milk-derived bioactive peptides on HCT-116 human colorectal carcinoma cell line

Novel food-derived anti cancerogenic bioactive peptides were characterized by goat milk pepsin hydrolysate. Pepsin treated casein fraction of goat milk caused an apoptotic cell death on the HCT116 cell lines. These bioactive peptides are encrypted in the protein structure in the inactive form and can become active during gastrointestinal digestion in the body. In this study, the possible therapeutic effect of goat milk-based bioactive peptides on human colorectal cancer cell lines was investigated. Goat milk-derived bioactive peptides were extracted from the casein and whey protein fractions using trypsin, pepsin, and papain enzymes. The bioactive peptides were characterized by the liquid chromatography quadrupole time of flight mass spectrometry. Both enzyme-treated casein and whey fractions were incubated with the HCT116 cell lines, and then the cell cytotoxicity was evaluated using MTT assay. The type of cell death was analyzed by flow cytometry using Annexin V and propidium iodide.Among all applications, the pepsin-treated casein fraction was the highest potential peptides that cause 80.92% apoptotic cell death. In conclusion, pepsin treated casein fraction exhibited antiproliferative activity against HCT116 cells. The bioactive peptides of this fraction can be considered as a potential source for the development of new anti cancerogenic agents.

Targeting oncogenic mutations in colorectal cancer using cryptotanshinone.

Colorectal cancer (CRC) is one of the most prevalent types of cancer in the world and ranks second in cancer deaths in the US. Despite the recent improvements in screening and treatment, the number of deaths associated with CRC is still very significant. The complexities involved in CRC therapy stem from multiple oncogenic mutations and crosstalk between abnormal pathways. This calls for using advanced molecular genetics to understand the underlying pathway interactions responsible for this cancer. In this paper, we construct the CRC pathway from the literature and using an existing public dataset on healthy vs tumor colon cells, we identify the genes and pathways that are mutated and are possibly responsible for the disease progression. We then introduce drugs in the CRC pathway, and using a boolean modeling technique, we deduce the drug combinations that produce maximum cell death. Our theoretical simulations demonstrate the effectiveness of Cryptotanshinone, a traditional Chinese herb derivative, achieved by targeting critical oncogenic mutations and enhancing cell death. Finally, we validate our theoretical results using wet lab experiments on HT29 and HCT116 human colorectal carcinoma cell lines.

Biotransformation of Food-Grade and Nanometric TiO2 in the Oral-Gastro-Intestinal Tract: Driving Forces and Effect on the Toxicity toward Intestinal Epithelial Cells.

Background: Oral exposure to titanium dioxide (TiO2) is common since it is widely used in food and pharmaceutical products. Concern on the safety of this substance has been recently raised, due to the presence of an ultrafine fraction in food-grade TiO2. Discrepancy exists among data reported in in vitro and in vivo studies on intestinal acute/chronic toxicity of TiO2. This might be due to the different biological identity of TiO2 in traditional in vitro test by respect in vivo conditions. Methods: One food-grade TiO2 and two nanometric TiO2 samples were treated with a simulated human digestive dystem (SHDS) in order to investigate the bio-transformation occurring to the particles once ingested in term of size distribution (Dynamic Light Scattering—DLS-, Flow Particle Imaging, Asymmetric Flow Field Flow Fractionation-AF4-) and surface modification (Electrophoretic Light Scattering—ELS-, Electron Paramagnetic Resonance Spectroscopy—EPR-). The effect of SHDS on the cyto-, genotoxicity and potential to induce oxidative stress towards human colorectal carcinoma HCT116 cells was also assessed. Results: Aggregation as a consequence of the high ionic strength of the gastric and intestinal simulated fluids was observed, together with the formation of a partially irreversible bio-corona containing phosphate ions and proteins. Such bio-corona led to a partial masking of the TiO2 particles surface and reactivity. Pristine and treated TiO2 nanoparticles showed comparable acute toxicity and genotoxicity toward HCT116 cells, whereas a small decrease of the induction of oxidative stress after treatment was observed. Conclusions: Overall the results underline the importance of SHDS as a tool to improve the predictive power of in vitro tests towards intestinal nanomaterial toxicity.

Olaparib Synergizes the Anticancer Activity of Daunorubicin via Interaction with AKR1C3.

Olaparib is a potent poly (ADP-ribose) polymerase inhibitor currently used in targeted therapy for treating cancer cells with BRCA mutations. Here we investigate the possible interference of olaparib with daunorubicin (Daun) metabolism, mediated by carbonyl-reducing enzymes (CREs), which play a significant role in the resistance of cancer cells to anthracyclines. Incubation experiments with the most active recombinant CREs showed that olaparib is a potent inhibitor of the aldo-keto reductase 1C3 (AKR1C3) enzyme. Subsequent inhibitory assays in the AKR1C3-overexpressing cellular model transfected human colorectal carcinoma HCT116 cells, demonstrating that olaparib significantly inhibits AKR1C3 at the intracellular level. Consequently, molecular docking studies have supported these findings and identified the possible molecular background of the interaction. Drug combination experiments in HCT116, human liver carcinoma HepG2, and leukemic KG1α cell lines showed that this observed interaction can be exploited for the synergistic enhancement of Daun's antiproliferative effect. Finally, we showed that olaparib had no significant effect on the mRNA expression of AKR1C3 in HepG2 and KG1α cells. In conclusion, our data demonstrate that olaparib interferes with anthracycline metabolism, and suggest that this phenomenon might be utilized for combating anthracycline resistance.

Reactivity and Anticancer Assessment of 4-Hydroxyquinoline Derivatives

Ethyl 4-hydroxy-7-(trifluoromethyl)quinoline-3-carboxylate (1) exhibits a moderate cytotoxic activity against the MCF-7 mammary gland cancer cell line and HePG2 hepatocellular carcinoma cell line and a weak activity against the HCT-116 human colorectal carcinoma cell line. In order to enhance the cytotoxic activity of this compound, it was modified by changing the side-chain substituent and/or forming a new heterocyclic ring fused to the pyridine ring. Heating compound 1 with chloroacetyl chloride gave a mixture of two isomeric O-acylation products ethyl 4-(2-chloroacetoxy)-7-(trifluoromethyl)-quinoline-3-carboxylate and 3-chloro-8-(trifluoromethyl)-2H-pyrano[3,2-c]quinoline-2,4(3H)-dione, whereas the reaction with acetyl chloride in NaOH formed an N-acylation product 1-acetyl-4-oxo-7-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid. The reactions of compound 1 with urea, thiourea, hydrazine hydrate, hydroxylamine, o-phenylenediamine, phenyl isothiocyanate, and ethyl acetoacetate yielded the corresponding condensation products 1-[4-oxo-7-(trifluoromethyl)-3,4-dihydroquinoline-3-carbonyl)]urea, 1-[4-oxo-7-(trifluoromethyl)-3,4-dihydroquinoline-3-carbonyl)]thiourea, 7-(trifluoromethyl)-1,2-dihydropyrazolo[4,3-c]quinolin-3-one, 7-(trifluoromethyl)isoxazolo[4,5-c]quinolin-3(2H)-one, 3-(trifluoromethyl)-13H-benzo[2,3][1,4]diazepino[6,5-c]quinolin-7-ol, 3-phenyl-2-thioxo-8-(trifluoromethyl)-2,3-dihydro-4H-[1,3]oxazino[5,6-c]quinolin-4-one and ethyl 8-(trifluoromethyl)-2-methyl-4-oxo-4H-pyrano[3,2-c]quinolone 3-carboxylate, respectively. The structures of the synthesized compounds were confirmed by elemental analysis and spectral data.

In Vitro Evaluation of Apoptotic Induction of Butyric Acid Derivatives in Colorectal Carcinoma Cells.

Butyric acid, a short chain fatty acid, plays an important role in the prevention of colon cancer. The aim of this study was to analyze the growth inhibitory and apoptotic effect of butyric acid derivatives in colorectal cancer cells. Human colorectal carcinoma HCT116 cells, were treated with the IC50 concentration of sodium butyrate, indole-3-butyric acid, tributyrin and 2-amino-n-butyric acid. Comet assay, caspase-3 assay and cell-cycle analysis were used to analyze apoptosis. Tributyrin and indole-3-butyric acid showed the least IC50 values at 24 h incubation. Butyric acid derivatives significantly activated caspase-3 activity compared with the control. Additionally, indole-3-butyric acid and tributyrin caused G0/G1 and G2/M phase arrest. Butyric acid derivatives effectively induced apoptosis in HCT116 cells.

Abstract 707: Emerging role of mitochondrial pathway in crosstalk between ferroptosis and apoptosis

Abstract Introduction: Several processes lead to cell death; ferroptosis, apoptosis, necrosis, and autophagy are the primary mechanisms. Each type of biological death has been believed to have a distinct biochemical and morphological fingerprint. However, emerging evidence suggests that these different types of cell death often share common pathways. In this study, we investigated whether mitochondrial signaling pathways play an important role in the intracellular communication between ferroptosis and apoptosis. Methods: The human colorectal carcinoma HCT116 cell line was used to evaluate the interplay between cell death pathways. Cells were treated with the apoptotic agent TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and ferroptotic agents such as artesunate and erastin. Cell death was determined by trypan blue exclusion assay. Apoptosis was measured by western blot. Ferroptosis was assessed using MDA assay. Bax oligomerization was performed by mitochondrial fractionation followed by SDS-PAGE under nondenaturing conditions. Results: We observed that the combinatorial treatment induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed an increase in pro-apoptotic Bax oligomerization in the mitochondrial fraction. The combined treatment-induced apoptosis was suppressed in Bax-deficient HCT116 cells. Ferroptotic agents enhanced the intracellular level of anti-apoptotic Mcl-1. However, the combined treatment decreased the level of Mcl-1. Interestingly, the combined treatment-induced apoptosis was abolished in HCT116 cells with knockin of Mcl-1 phosphorylation site mutant (S121A/E125A/S159A/T163A). Conclusions: These data suggest a novel mechanism for the combined treatment of the apoptotic agent TRAIL and ferroptotic agent erastin/artesunate-induced synergistic apoptosis through the mitochondrial pathway. Citation Format: Yong J. Lee. Emerging role of mitochondrial pathway in crosstalk between ferroptosis and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 707.

Luteolin Shifts Oxaliplatin-Induced Cell Cycle Arrest at G₀/G₁ to Apoptosis in HCT116 Human Colorectal Carcinoma Cells.

Certain antioxidative flavonoids are known to activate nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates cellular antioxidants and detoxifying response and is reportedly highly activated in many types of cancers. Few studies on the potential undesired effects of flavonoid intake during chemotherapy have been conducted, yet Nrf2 activators could favor cancer cell survival by attenuating chemotherapeutic efficiency. This study aimed to examine if luteolin, an Nrf2 activator, hinders chemotherapeutic activity of oxaliplatin, a potent anticancer agent for colorectal cancer, in HCT116 cells. Luteolin treatment strongly increased the transcriptional activity of the antioxidant response element in HCT116 cells and induced the protein expression of heme oxygenase-1, which were indicative of its Nrf2-inducing potential. Intriguingly, 25 μM luteolin reduced cell viability through apoptotic induction, which was intensified in p53-expressing cells while 1 μM oxaliplatin caused cell cycle arrest at G0/G1-phase via the p53/p21-dependent mechanism. Moreover, luteolin treatment was found to reduce oxaliplatin-treated p53-null cell viability and colony counts further, thereby demonstrating an additional effect of luteolin in the killing of human colorectal tumor HCT116 cells not expressing functional p53 protein. The findings suggest that luteolin can induce p53-mediated apoptosis regardless of oxaliplatin treatment and may eliminate oxaliplatin-resistant p53-null colorectal cells.

Adverse Effect of Luteolin on the Anticancer Ability of Oxaliplatin in HCT116 Human Colorectal Carcinoma Cells

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in the world. Intracellular oxidative stress by accumulation of reactive oxygen species (ROS) can cause redox imbalance that is associated with higher risks of cancer development. Dietary flavonoids activating a nuclear factor E2‐related factor 2/antioxidant response element (Nrf2/ARE) signaling pathway have received a lot of attention due to their abilities to reduce oxidative stress by expressing antioxidant/phase II detoxifying enzymes. Hence, some cancer patients taking chemotherapeutic agents have been consuming flavonoid‐rich food, expecting their anticancer efficacies to be synergistically enhanced. However, careful consideration of possible undesired effects of flavonoid intake during chemotherapy is needed; flavonoids activating Nrf2/ARE signaling pathway could favor cancer cell survival by attenuating ROS‐induced oxidative stress elevated by anticancer agents. The purpose of the present study was to examine how luteolin would affect chemotherapeutic activity of oxaliplatin, a commonly used anticancer agent for colon cancer, in HCT116 p53+/+ cell line and its derivative HCT116 p53−/− cells. Results showed that luteolin increased ARE‐luciferase activity, decreased ROS level by Nrf2/ARE activation and subsequently stimulated proliferation of oxaliplatin‐treated colorectal cancer cells. In addition, oxaliplatin and a high dose of luteolin induced cell cycle arrest and apoptosis in a p53‐dependent manner, respectively. However, oxaliplatin‐induced cell cycle arrest was compromised by the high dose of luteolin in HCT116 p53+/+ cells. Moreover, the high dose of luteolin suppressed oxaliplatin‐induced p21 expression in HCT116 p53+/+ cells, but not in HCT116 p53−/− cells. Current observations indicate that a high dose of luteolin can negatively affect oxaliplatin‐based chemotherapy in a p53‐dependent fashion, suggesting that Nrf2‐activating flavonoids may interfere with chemotherapeutic efficacy of anticancer agents in colorectal cancer cells that carry functional p53 protein. Further studies about its in vivo impact of luteolin on oxaliplatin‐based chemotherapy are needed to ascertain current findings.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT), Republic of Korea.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel Small Molecule p53 Stabilizer for Brain Cell Differentiation.

Brain tumor, as any type of cancer, is assumed to be sustained by a small subpopulation of stem-like cells with distinctive properties that allow them to survive conventional therapies and drive tumor recurrence. Thus, the identification of new molecules capable of controlling stemness properties may be key in developing effective therapeutic strategies for cancer by inducing stem-like cells differentiation. Spiropyrazoline oxindoles have previously been shown to induce apoptosis and cell cycle arrest, as well as upregulate p53 steady-state levels, while decreasing its main inhibitor MDM2 in the HCT116 human colorectal carcinoma cell line. In this study, we made modifications in this scaffold by including combinations of different substituents in the pyrazoline ring in order to obtain novel small molecules that could modulate p53 activity and act as differentiation inducer agents. The antiproliferative activity of the synthesized compounds was assessed using the isogenic pair of HCT116 cell lines differing in the presence or absence of the p53 gene. Among the tested spirooxindoles, spiropyrazoline oxindole 1a was selective against the cancer cell line expressing wild-type p53 and presented low cytotoxicity. This small molecule induced neural stem cell (NSC) differentiation through reduced SOX2 (marker of multipotency) and increased βIII-tubulin (marker of neural differentiation) which suggests a great potential as a non-toxic inducer of cell differentiation. More importantly, in glioma cancer cells (GL-261), compound 1a reduced stemness, by decreasing SOX2 protein levels, while also promoting chemotherapy sensitization. These results highlight the potential of p53 modulators for brain cell differentiation, with spirooxindole 1a representing a promising lead molecule for the development of new brain antitumor drugs.

Cytotoxic activity of in vitro Bacopamonnieri different extracts

Bacopamonnieri (BM) which is commonly known as “Brahmi,” belongs to the family Scrophulariaceae, (BM) is an herbaceous plant traditionally used from time immemorial in Ayurvedic and folklore medicines, found throughout the Indian subcontinent in wet, damp and marshy areas. Tissue culture studies were performed on Bacopamonnieri (Brahmi). Nodal explants containing preformed axillary buds were used as the starting material. Sterilized explants were aseptically cultured on MS medium supplemented with different plant growth regulators.This study aimed at evaluating the anticancer activity of extraction of (BM) with different solvents according to polarity, namely (methanol, chloroform, and acetone) against two different cell lines of colorectal cancer. Cell cytotoxicity effects of each extract of (BM)was carried out by means of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) cell viability assay using (human colorectal carcinoma) hct116 and (human epithelial colorectal adenocarcinoma) caco2 cell lines. The acetone extract showed the potent cytotoxicity at 48 hr (IC50 6.79 –6.1 μg/Ml) for hct116 and Caco2, respectively.

Cordycepin inhibits lipopolysaccharide-induced cell migration and invasion in human colorectal carcinoma HCT-116 cells through down-regulation of prostaglandin E2receptor EP4

Prostaglandin E2 (PGE2), a major product of cyclooxygenase-2 (COX-2), plays an important role in the carcinogenesis of many solid tumors, including colorectal cancer. Because PGE2 functions by signaling through PGE2 receptors (EPs), which regulate tumor cell growth, invasion, and migration, there has been a growing amount of interest in the therapeutic potential of targeting EPs. In the present study, we investigated the role of EP4 on the effectiveness of cordycepin in inhibiting the migration and invasion of HCT116 human colorectal carcinoma cells. Our data indicate that cordycepin suppressed lipopolysaccharide (LPS)-enhanced cell migration and invasion through the inactivation of matrix metalloproteinase (MMP)-9 as well as the down-regulation of COX-2 expression and PGE2 production. These events were shown to be associated with the inactivation of EP4 and activation of AMP-activated protein kinase (AMPK). Moreover, the EP4 antagonist AH23848 prevented LPS-induced MMP-9 expression and cell invasion in HCT116 cells. However, the AMPK inhibitor, compound C, as well as AMPK knockdown via siRNA, attenuated the cordycepin-induced inhibition of EP4 expression. Cordycepin treatment also reduced the activation of CREB. These findings indicate that cordycepin suppresses the migration and invasion of HCT116 cells through modulating EP4 expression and the AMPK-CREB signaling pathway. Therefore, cordycepin has the potential to serve as a potent anti-cancer agent in therapeutic strategies against colorectal cancer metastasis.

Serine/threonine kinase 31 promotes PDCD5-mediated apoptosis in p53-dependent human colon cancer cells.

Although programed cell death 5 (PDCD5) is an important protein in p53-mediated proapoptotic signaling, very little is known about PDCD5-related cell death. In this study, we report that serine/threonine kinase 31 (STK31) interacts with PDCD5, which maintains the stability of PDCD5. STK31 overexpression significantly activated PDCD5 stabilization and p53-mediated apoptosis in response to etoposide (ET). However, STK31 knockdown did not enhance apoptosis by ET treatment. Moreover, when STK31 was depleted, PDCD5 inhibited the activation of the p53 signaling pathway with ET, indicating that the PDCD5-STK31 network has an essential role in p53 activation. Importantly, STK31 activated the p53 signaling pathway by genotoxic stress through positive regulation of PDCD5-mediated apoptosis. We thus demonstrated that overexpression of STK31 greatly inhibited tumorigenic growth and increased the chemosensitivity of HCT116 human colorectal carcinoma cells. Taken together, these findings demonstrate that the STK31-PDCD5 complex network regulates apoptosis of cancer cells, and STK31 is a positive apoptosis regulator that inhibits tumorigenesis of colon cancer cells by inducing PDCD5-mediated apoptosis in response to genotoxic stress.

Spectroscopic and theoretical investigation of the potential anti-tumor and anti-microbial agent, 3-(1-((2-hydroxyphenyl)amino)ethylidene)chroman-2,4-dione.

The coumarin-orthoaminophenol derivative was prepared under mild conditions. Based on crystallographic structure, IR and Raman, 1H and 13C NMR spectra the most applicable theoretical method was determined to be B3LYP-D3BJ. The stability and reactivity parameters were calculated, in the framework of NBO, QTAIM and Fukui functions, form the optimized structure. This reactivity was then probed in biological systems. The antimicrobial activity towards four bacteria and three fungi species was examined and activity was proven. In vitro cytotoxic effects, against human epithelial colorectal carcinoma HCT-116 and human healthy lung MRC-5 cell lines, of the investigated substance are also tested. Compound showed significant cytotoxic effects on HCT-116 cells, while on MRC-5 cells showed no cytotoxic effects. The effect of hydroxy group in ortho-position on the overall reactivity of molecule was examined through molecular docking with Glutathione-S-transferases.

Doped ZnO nanoparticles impregnated on Kaolinite (Clay): A reusable nanocomposite for photocatalytic disinfection of multidrug resistant Enterobacter sp. under visible light

Water contamination by multidrug resistant (MDR) enteric bacteria can be considered as the foremost cause of gastrointestinal infections and poses a threat to global public health. Therefore, there is an urgent need to pursue unorthodox techniques with potential of community scale applications for purging of water borne pathogenic bacteria. We communicate visible-light assisted photocatalytic disinfection (PCD) of an enteric MDR bacterium; Enterobacter sp. using Fe-doped ZnO nanoparticles impregnated on Kaolinite (Clay) (ZnO/K). ZnO/K was synthesized by co-precipitation technique and was found to be more effective than Fe-doped ZnO (ZnO) and Kaolinite for PCD process. Analysis from fluorescence microscopy and electron microscopy (FESEM) proposed complete bacterial cell death via PCD due to damage of bacterial cell membrane. Experimental evidences indicated that O2− could be acting as the most significant component in disinfection of MDR Enterobacter sp. in visible-light assisted PCD process in presence of ZnO/K. Considering the experimental data of Resazurin assay, it is proposed that reactive oxygen species (ROS) generated during PCD might have impeded the oxido-reductase enzyme system of the bacteria and hence trammeling its metabolic activity. Crystal structure and particle size of ZnO/K was found to be unaltered during the photocatalytic process indicating its potential for reusability. When ZnO/K was exposed to HCT-116 Human Colorectal Carcinoma cell lines, about 79% cell survivability was noticed. The synthesized material was successful in completely disinfecting the target microorganism in Zebra Fish model, without producing any adverse effects on the Fish itself, further reinforcing its biocompatibility factor. High effectiveness of PCD process using ZnO/K under visible light in disinfecting enteric MDR bacteria, might have promising outcome as an alternative water disinfection technology to prevent the spread of infectious and resistant bacteria without producing any adverse effect on non-specific flora and fauna.

Mitochondrial dysfunction suppresses p53 expression via calcium-mediated nuclear factor-kB signaling in HCT116 human colorectal carcinoma cells.

Mitochondrial DNA (mtDNA) mutations are often observed in various cancer types. Although the correlation between mitochondrial dysfunction and cancer malignancy has been demonstrated by several studies, further research is required to elucidate the molecular mechanisms underlying accelerated tumor development and progression due to mitochondrial mutations. We generated an mtDNA-depleted cell line, ρ0, via long-term ethidium bromide treatment to define the molecular mechanisms of tumor malignancy induced by mitochondrial dysfunction. Mitochondrial dysfunction in ρ0 cells reduced drug-induced cell death and decreased the expression of pro-apoptotic proteins including p53. The p53 expression was reduced by activation of nuclear factor-κB that depended on elevated levels of free calcium in HCT116/ρ0 cells. Overall, these data provide a novel mechanism for tumor development and drug resistance due to mitochondrial dysfunction.

Dioscorea bulbifera induced apoptosis through inhibition of ERK 1/2 and activation of JNK signaling pathways in HCT116 human colorectal carcinoma cells

Dioscorea bulbifera, also known as air potato, has been cultivated as food crop mainly in tropical countries in Asia and Australia. The tubers are edible and have often been used in Traditional Chinese Medicine (TCM) and Ayurvedic medicine to treat cancer, diabetes, thyroid disease, and inflammation. This study aimed to investigate the effects of D. bulbifera on HCT116 human colorectal carcinoma cells and to unravel the plausible mechanisms underlying its apoptotic effects. The ethanol crude and fractions (hexane, ethyl acetate and water) of D. bulbifera were subjected to cell viability MTT assay against various cancer cell lines. The lowest IC50 of the extract and fractions on selected cancer cells were selected for further apoptosis assay and western blot analysis. HCT116 cancer cells were treated with D. bulbifera and stained with Annexin/PI or Hoechst 33342/PI for preliminary confirmation of apoptosis. The dissipation of mitochondria membrane potential (MMP) was determined by flow cytometry. The protein expressions of apoptosis-related proteins such as Bcl-2 family, caspases, Fas, PARP, ERK1/2 and JNK were detected by western blot analysis. Moreover, the HCT116 cells were treated with UO126 and SP600125 inhibitors to verify the involvement of ERK1/2 and JNK protein expressions in inducing apoptotic cell death. Based on the result, D. bulbifera ethyl acetate fraction (DBEAF) exhibited the most compelling cytotoxicity on HCT116 cells with an IC50 of 37.91 ± 1.30 µg/mL. The induction of apoptosis was confirmed by phosphatidylserine externalization and chromatin condensation. Depolarization of MMP further conferred the induction of apoptosis was through the regulation of Bcl-2 family proteins. Activation of caspase cascades (caspase-3, -9, -8 and -10) was elicited followed by the observation of cleaved PARP accumulation in DBEAF-treated cells. Furthermore, death receptor, Fas was activated upon exposure to DBEAF. Collective apoptotic evidences suggested the involvement of intrinsic and extrinsic pathways by DBEAF in HCT116 cells. Interestingly, the attenuation of ERK1/2 phosphorylation accompanied by the activation of JNK was detected in DBEAF-treated cells. In conclusion, the findings revealed that DBEAF induced apoptosis through intrinsic and extrinsic pathways involving ERK1/2 and JNK.

New histone demethylase LSD1 inhibitor selectively targets teratocarcinoma and embryonic carcinoma cells

LSD1/KDM1 is a histone demethylase that preferentially removes methyl groups from the mono- and di-methylated lysine 4 in histone H3 (H3K4), key marks for active chromatin for transcriptional activation. LSD1 is essential for pluripotent embryonic stem cells and embryonic teratocarcinoma/carcinoma cells and its expression is often elevated in various cancers. We developed a new LSD1 inhibitor, CBB3001, which potently inhibited LSD1 activity both in vitro and in vivo. CBB3001 also selectively inhibited the growth of human ovarian teratocarcinoma PA-1 and mouse embryonic carcinoma F9 cells, caused the downregulation of pluripotent stem cell proteins SOX2 and OCT4. However, CBB3001 does not have significant inhibition on the growth of human colorectal carcinoma HCT116 cells or mouse fibroblast NIH3T3 cells that do not express these stem cell proteins. Our studies strongly indicate that CBB3001 is a specific LSD1 inhibitor that selectively inhibits teratocarcinoma and embryonic carcinoma cells that express SOX2 and OCT4.

Grape seed proanthocyanidins induce mitochondrial pathway-mediated apoptosis in human colorectal carcinoma cells.

Grape seed proanthocyanidins (GSPs) have been reported to possess a wide array of pharmacological and biochemical properties. Recently, GSPs have been reported to inhibit various types of colorectal cancer; however, the mechanism(s) involved remain unclear. The present study investigated the effects of GSPs on HCT-116 human colorectal carcinoma cell line. Exposure of these cells to GSPs for 48 h resulted in a significant concentration-dependent inhibition of cell viability. Further investigation indicated that GSPs induced apoptosis of these cells. Analyses of mRNA expression levels using reverse transcription-quantitative polymerase chain reaction and protein expression levels by western blotting revealed that this was associated with increased expression levels of p53 tumor suppressor protein, cytochrome c, and pro-apoptotic proteins, apoptosis regulator Bax (Bax) and Bcl-2 homologous antagonist/killer. Furthermore, decreased expression levels of the anti-apoptotic protein, B cell lymphoma-2 and activation of caspase-2, caspase-3 and caspase-9 were demonstrated. GSP-induced loss of mitochondrial membrane potential was also detected by JC-1 assay. These findings suggested that GSPs induced colon cancer cell apoptosis via the mitochondrial signaling pathway. This provided evidence indicating that GSPs may provide potential chemotherapeutic agents for colorectal cancer.