Inhibited HT-29 Cell Proliferation Research Articles

Anticancer potential of fermented milk with autochthonous lactic acid bacteria.

The purpose of this study was to investigate the effect of fermented milk supernatants of autochthonous lactic acid bacteria, including Lactobacillus helveticus KMCH1 (ON561781), Lactococcus lactis KMCM3 (ON561782), and Lactiplantibacillus plantarum KMJC4 (ON615217), on human colon cancer (HT-29) and normal mouse fibroblast (L929) cells in vitro. Proteolytic activity, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test, evaluation of apoptosis induction, and cell cycle arrest by flow cytometry were the assays performed in this study. The measurement of proteolytic activity of three types of fermented milk supernatant using an orthophthalaldehyde reagent showed that the fermented milk supernatant of L. helveticus KMCH1 included the highest proteolysis. Three types of fermented milk supernatant showed anticancer effects on HT-29 cell in a time- and concentration-based manner (at a concentration of 16 mg ml-1 for 72 h of incubation), while the effect of three types of supernatant on inhibition of L929 cell was 3%-10%. Besides, three types of supernatant inhibited HT-29 cell proliferation by inducing apoptosis and cell cycle arrest in the S phase. Autochthonous lactic acid bacteria strains were able to produce bioactive peptides with anticancer effects in fermented milk. Inhibition of HT-29 cell proliferation was dependent on peptide concentration.

A free fatty acid receptor agonist inducing autophagy in HT-29 cells by downregulating the AKT/mTOR signaling pathway.

GW9508, a free fatty acid receptor agonist acts in a G-coupled protein receptor 40 (GPR40)-dependent pathway. Here, we investigated the induction of stress oxidative and autophagy by GW9508 in the human colorectal cancer cell line (HT-29) and the crosstalk between autophagy and apoptotic in HT-29 cells. HT-29 was treated with GW9508 at a concentrations range of 50-500 μM in fibrin gel. Cell viability was investigated using an MTT assay. Induction of autophagy and apoptosis was assessed through Western blotting for associated proteins, acridine orange staining, MDC staining, qRT-PCR, and electron microscopy. Also, we estimated the molecular interactions between GW9805 and some markers through molecular docking. GW9508 inhibited HT-29 cell proliferation, induced apoptosis, and resulted in autophagy. The induced autophagy in cells was confirmed by the observation of autophagosomes, the presence of autophagy markers, including beclin-1, LC3, AMPK, and lack expression of mTOR and AKT. Moreover, GW9508 treatment significantly increased the expression of catalase and superoxide dismutase in cells. Our results indicated that GW9508 could induce autophagy by inhibiting the Akt/mTOR in HT-29. Hence, GW9508 is suggested as a novel anticancer reagent.

Investigation of the effects of isoeugenol-based phenolic compounds on migration and proliferation of HT29 colon cancer cells at cellular and molecular level

Colorectal cancer is a type of cancer encountered worldwide and ranks third among all cancer types in terms of incidence. Polyphenols have been shown to have a wide range of biological functions, including a significant impact on cancer start, development, and promotion through regulating many signaling pathways. The aim of this study was to investigate the anticancer effects of isoeugenol based compounds 1, 2 on HT29 colorectal cancer cell line in vitro. MTT test and scratch assay were carried out to determine the effect of these compounds on HT29 cell proliferation and migration respectively. In addition, mRNA expression levels of apoptosis and metastasis-related genes (p53, Bcl2, Bax, Caspase 3, Caspase7, Caspase8, Caspase9, HIF1-α, VEGF, MMP-2, MMP-9) were examined by quantitative real-time PCR. The results indicated that 1 and 2 inhibited HT29 cell proliferation and induced apoptosis by increasing the Bax/Bcl2 ratio and Caspase-9 and Caspase-3 mRNA expression. In conclusion, the results of this study showed that the treatment of these compounds significantly suppressed the mRNA expressions of metastasis-related genes such as Matrix Metalloproteinase-2, Matrix Metalloproteinase-9, Vascular Endothelial Growth Factor and Hypoxia‑Inducible Factor 1α.

The Cell Cycle-Associated Protein CDKN2A May Promotes Colorectal Cancer Cell Metastasis by Inducing Epithelial-Mesenchymal Transition.

Colorectal cancer (CRC) is a common gastrointestinal malignancy, and recurrence and metastasis contribute considerably to its high mortality. It is well known that the epithelial-mesenchymal transition (EMT) accelerates the rate of cancer cell dissemination and migration, thus promoting cancer metastasis. Targeted therapy is a common modality for cancer treatment, and it can play a role in inhibiting cancer progression. In this study, bioinformatics was used to search for genes associated with the prognosis of CRC. First, differential analysis was performed on colon and rectal cancer samples to obtain 2,840 and 3,177 differentially expressed genes (DEGs), respectively. A Venn diagram was then used to identify 262 overlapping genes from the two groups of DEGs and EMT-related genes. The overlapping genes were subjected to batch survival analysis and batch expression analysis successively, and nine genes were obtained whose high expression in CRC led to a poor prognosis. The least absolute shrinkage and selection operator (LASSO) prognostic model was then constructed to obtain the risk score formula. A nomogram was constructed to seek prognostic independent factors to obtain CDKN2A. Finally, CCK-8 assay, flow cytometry and western blotting assays were performed to analyze the cellular biological function of CDKN2A. The results showed that knockdown of CDKN2A expression inhibited HT-29 cell proliferation, promoted apoptosis and cell cycle progression, and affected the EMT process in CRC.

Luteolin induces mitochondrial apoptosis in HT29 cells by inhibiting the Nrf2/ARE signaling pathway.

The aim of the current study was to investigate luteolin-induced apoptosis and the molecular mechanisms underlying it in HT29 cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the cytotoxicity of luteolin on HT29 cells, and a dichloro-dihydro-fluorescein diacetate assay was used to measure cellular levels of reactive oxygen species (ROS). The effects of luteolin on the mitochondrial membrane potential were also evaluated. Bax and Bcl-2 mRNA expression were determined using reverse transcription-quantitative PCR. Additionally, western blot analysis was performed to assess changes in cytochrome c and caspase-3 protein expression. Localization of nuclear factor erythroid 2-related factor 2 (Nrf2) in the nucleus was also assessed using immunofluorescence. Luteolin exhibited cytotoxicity on HT29 cells in a time- and concentration-dependent manner. Additionally, ROS production was indicated to be increased and ROS scavenging was decreased, which resulted in a significant increase in the levels of ROS in the cells. The mitochondrial membrane potential was indicated to decrease following luteolin treatment. At the molecular level, luteolin significantly increased the mRNA expression of Bax and the protein expression of cytochrome c, caspase-3, p47phox and p22phox. The results revealed that luteolin decreased Bcl-2 protein expression and inhibited the nuclear localization of Nrf2. In conclusion, the current study indicated that luteolin inhibited HT29 cell proliferation and induced apoptosis via the mitochondrial pathway.

Parthenolide inhibits transforming growth factor β1-induced epithelial-mesenchymal transition in colorectal cancer cells.

Background/AimsTransforming growth factor-β1 (TGF-β1) induction of epithelial-mesenchymal transition (EMT) is one of the mechanisms by which colorectal cancer (CRC) cells acquire migratory and invasive capacities, and subsequently metastasize. Parthenolide (PT) expresses multiple anti-cancer and anti-inflammatory activities that inhibit nuclear factor κB by targeting the IκB kinase complex. In the present study, we aimed to investigate whether PT can inhibit TGF-β1-induced EMT in CRC cell lines.MethodsHT-29 and SW480 cell lines were used in the experiment. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and sub-G1 analysis was measured by flow cytometry. The induction of EMT by TGF-β1 and inhibition of the process by PT was analyzed by phase contrast microscopy, wounding healing, cellular migration and invasion assays, and Western blotting.ResultsTGF-β1 inhibits HT-29 cell proliferation, but has no effect on SW480 cell proliferation; different concentrations of TGF-β1 did not induce apoptosis in HT-29 and SW480 cells. PT attenuates TGF-β1-induced elongated, fibroblast-like shape changing in cells. PT inhibits TGF-β1-induced cell migration and cell invasion. In addition, other EMT markers such as β-catenin, Vimentin, Snail, and Slug were suppressed by PT, while E-cadherin was increased by PT.ConclusionsOur findings show that PT inhibits TGF-β1-induced EMT by suppressing the expression of the mesenchymal protein and increasing expression of the epithelial protein. These findings suggest a novel approach for CRC treatment by suppression of TGF-β1-induced EMT.

Mycophenolate Mofetil Alone and in Combination with Tacrolimus Inhibits the Proliferation of HT-29 Human Colonic Adenocarcinoma Cell Line and Might Interfere with Colonic Tumorigenesis.

Familial adenomatous polyposis (FAP) was found to be completely reversed in a patient treated with mycophenolate mofetil (MMF) and tacrolimus following kidney transplantation. In this preliminary study, we assessed whether MMF and tacrolimus alone or in combination interfere with the cell cycle and proliferation in a human colonic adenocarcinoma cell line and in the colonic polyps of the patient with FAP. Human colonic adenocarcinoma HT-29 cells were treated with tacrolimus and MMF alone and in combination at different concentrations. Cell viability and proliferation were assessed using the MTT assay. Cell-cycle distribution was analyzed by flow cytometry. Expression of Ki-67, a marker of mitotic activity, was evaluated in the patient's colonic polyps before and under drug treatment. MMF in combination with tacrolimus induced S-phase cell-cycle arrest and markedly inhibited HT-29 cell proliferation. Ki-67 expression in the patient's colonic polyps was significantly reduced following combined tacrolimus and MMF treatment. MMF and tacrolimus synergistically inhibited proliferation of a human colonic adenocarcinoma cell line and interfered with the expansion of colonic crypt proliferation in the polyp from a patient with FAP. The results confirm our clinical observation and indicate the possibility of novel approach to therapy of colorectal neoplasia.

Silencing protein kinase C ζ by microRNA-25-5p activates AMPK signaling and inhibits colorectal cancer cell proliferation.

Developing novel strategies against human colorectal cancer (CRC) cells is needed. Activation of AMP-activated protein kinase (AMPK) could possibly inhibit CRC cells. Protein kinase C ζ (PKCζ) is an AMPK negative regulator. Here we found that PKCζ expression was significantly elevated in human colon cancer tissues and CRC cells. PKCζ upregulation was correlated with AMPK in-activation and mTOR complex 1 (mTORC1) over-activation. Reversely, PKCζ shRNA knockdown activated AMPK signaling and inhibited HT-29 cell proliferation. Significantly, downregulation of microRNA-25-5p (miR-25-5p), a PKCζ-targeting miRNA, could be the cause of PKCζ upregulation. Exogenous expression of miR-25-5p silenced PKCζ to activate AMPK signaling, which inhibited HT-29 cell proliferation. In vivo studies showed that HT-29 xenograft growth in mice was inhibited after expressing PKCζ shRNA or miR-25-5p. Collectively, PKCζ could be a novel oncogenic protein of human CRC. PKCζ silence, by targeted-shRNA or miR-25-5p expression, activates AMPK and inhibits HT-29 cell proliferation.

Treatment mechanism of matrine in combination with irinotecan for colon cancer.

The inhibitory effect of matrine (MA) was studied in combination with irinotecan (CPT-11) on proliferation of human colon carcinoma cell line HT29. We also explored the mechanism of cell apoptosis induction in HT29. HT29 cells were treated with different concentrations of MA and CPT-11 alone and in combination. The growth inhibition in HT29 cells was evaluated using MTT assay. Apoptosis was detected using AV-PI double staining flow cytometry. Transmission electron microscopy was used to detect structural changes in cells. Topoisomerase (TOPO) I, Bax and Caspase-3 expression levels were evaluated using western blot analysis. MA and CPT-11 alone and in combination, inhibited the proliferation of HT29 cells, whereas the combination treatment exhibited higher inhibitory effect (P<0.01). This suggests the existence of synergistic cytotoxicity. Compared with each treatment alone, the combination treatment caused more significant damage to cell structure, and caused a significantly higher apoptosis rate (P<0.01). Additionally, the combination treatment increased TOPO I, Bax and Caspase-3 expression levels (P<0.01). In conclusion, MA in combination with CPT-11 synergistically inhibited HT29 cell proliferation and induced apoptosis in these cells. The mechanism may be related to upregulation of the TOPO I, Bax and Caspase-3 protein expression.

Celastrus orbiculatus extract triggers apoptosis and autophagy via PI3K/Akt/mTOR inhibition in human colorectal cancer cells.

Celastrus orbiculatus is used as a folk medicine in China for the treatment of numerous diseases. The ethyl acetate extract of Celastrus orbiculatus (COE) also displays a wide range of anti-cancer activities in the laboratory. However, the effectiveness of COE-induced autophagy and its mechanism of action in colorectal cancer cells have not been investigated thus far. The present study analyzed the effect of COE on HT-29 cell viability, apoptosis and autophagy using MTT assay, flow cytometry, transmission electron microscopy and western blotting. Additionally, the autophagy inhibitor 3-methyladenine and the autophagy inducer rapamycin were used to further explore the effects of COE-induced autophagy in HT-29 cells. The present study also examined whether the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K) signaling pathway was involved in the regulation of COE-induced autophagy. The results revealed that COE inhibited HT-29 cell proliferation and decreased cell survival in a time- and dose-dependent manner, and that COE possessed the ability to induce both apoptosis and autophagy in HT-29 cells. Furthermore, autophagy and apoptosis induced by COE synergized to inhibit colorectal cancer growth. In addition, COE treatment decreased the phosphorylation of Akt and its downstream effectors mTOR and p70S6K. Taken together, these results demonstrate that both autophagy and apoptosis were activated during COE treatment of HT-29 cells, and that COE-induced autophagy decreases the viability of HT-29 cells via a mechanism that may depend on the PI3K/Akt/mTOR/p70S6K signaling pathway. Furthermore, compounds that induce autophagy administered in combination with COE may be an attractive strategy for enhancing the anti-tumor potency of COE in colorectal cancer.

Abstract 4619: Sirt2 protein is required for activation of mTOR signaling in colorectal cancer cells

Abstract The human sirtuin proteins (Sirt1-7) possess protein deacetylase activities and are involved in the regulation of metabolism, differentiation and cell survival. Sirt2 is upregulated in some cancers; however, some studies have suggested its role as that of a tumor suppressor. mTOR regulates many processes that control growth, including protein synthesis, oxidative metabolism, glucose homeostasis, cell growth, and lipogenesis. Deptor protein binds mTOR through the PDZ domain and inhibits mTOR activity. Our previous studies have shown that mTOR is a central regulator of colorectal cancer (CRC) cell proliferation and survival. The purpose of this study was to investigate: i) the regulation of Deptor expression by Sirt2, and ii) the role of Sirt2 protein in mTOR activation in CRC and normal intestinal cells. METHODS. Human CRC cell lines, HT29 and Caco-2, the rat normal intestinal cell line, IEC-6, and the human embryonic kidney cell line, HEK293, were transfected with siRNA targeting Sirt2 or control nontargeting siRNA. Cells were also infected with lentiviral vectors containing the control shRNA or shRNA to human Sirt2; stably expressing cells were selected with puromycin. Sirt2 overexpression was achieved by infecting cells with an adenovirus encoding Sirt2. Cell proliferation was assessed by counting the number of viable cells. The expression of Sirt2, Deptor, p-Akt (S473), Akt, p-S6 (S235/236), S6, LC3B, Cyclin D1, c-Myc, and survivin was analyzed by western blot. Deptor mRNA levels were determined by real time RT-PCR. RESULTS. Knockdown of Sirt2 inhibited mTOR signaling, decreased expression of cyclin D1, c-Myc, and survivin, and inhibited CRC cell growth. In agreement with the inhibition of mTOR activation, knockdown of Sirt2 significantly increased Deptor mRNA and protein expression in CRC cells. Conversely, overexpression of Sirt2 decreased Deptor expression and activated mTOR signaling. Knockdown of Sirt2 increased, while knockdown of Deptor decreased, the protein expression of LC3B, an autophagy marker, suggesting Sirt2 inhibition increases autophagy through induction of Deptor. Knockdown of Sirt2 inhibited HT29 cell proliferation; this inhibition was enhanced by treatment with 3MA, an autophagy inhibitor. In contrast, knockdown or overexpression of Sirt2 did not affect either the expression of Deptor or the activation of mTOR in HEK293 and IEC6 cells, suggesting a differential regulation of Deptor/mTOR signaling by Sirt2 in CRC and normal intestinal cells. CONCLUSIONS. We provide evidence that the Sirt2 protein increases mTOR activity and contributes to CRC cell proliferation. Importantly, our results indicate a novel role of Sirt2 in the activation of mTOR signaling in CRC cells but not in normal intestinal cells and suggests that targeted inhibition of Sirt2 could represent a novel and safe strategy for the treatment of CRC. Citation Format: Qingding Wang, Yuning Zhou, Heidi L. Weiss, B. Mark Evers. Sirt2 protein is required for activation of mTOR signaling in colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4619.

Recombinant Lactococcus lactis NZ9000 secretes a bioactive kisspeptin that inhibits proliferation and migration of human colon carcinoma HT-29 cells.

BackgroundProteinaceous bioactive substances and pharmaceuticals are most conveniently administered orally. However, the facing problems are the side effects of proteolytic degradation and denaturation in the gastrointestinal tract. In recent years, lactic acid bacteria (LAB) have been verified to be a promising delivery vector for susceptible functional proteins and drugs. KiSS-1 peptide, a cancer suppressor, plays a critical role in inhibiting cancer metastasis and its activity has been confirmed by direct administration. However, whether this peptide can be functionally expressed in LAB and exert activity on cancer cells, thus providing a potential alternative administration manner in the future, has not been demonstrated.ResultsA recombinant Lactococcus lactis strain NZ9000-401-kiss1 harboring a plasmid containing the gene of the tumor metastasis-inhibiting peptide KiSS1 was constructed. After optimization of the nisin induction conditions, the recombinant strain efficiently secreted KiSS1 with a maximum detectable amount of 27.9 μg/ml in Dulbecco’s Modified Eagle medium. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide and would healing assays, respectively, indicated that the secreted KiSS1 peptide remarkably inhibited HT-29 cell proliferation and migration. Furthermore, the expressed KiSS1 was shown to induce HT-29 cell morphological changes, apoptosis and reduce the expression of matrix metalloproteinase 9 (MMP-9) at both mRNA and protein levels.ConclusionsA recombinant L. lactis NZ9000-401-kiss1 successfully expressing the human kiss1 was constructed. The secreted KiSS1 peptide inhibited human HT-29 cells’ proliferation and migration probably by invoking, or mediating the cell-apoptosis pathway and by down regulating MMP-9 expression, respectively. Our results suggest that L. lactis is an ideal cell factory for secretory expression of tumor metastasis-inhibiting peptide KiSS1, and the KiSS1-producing L. lactis strain may serve as a new tool for cancer therapy in the future.

Molecular Mechanism of Enhanced Anticancer Effect of Nanoparticle Formulated LY2835219 via p16-CDK4/6-pRb Pathway in Colorectal Carcinoma Cell Line

LY2835219 is a dual inhibitor to CDK4 and CDK6. This study was to prepare LY2835219-loaded chitosan nanoparticles (CNP/LY) and LY2835219-loaded hyaluronic acid-conjugated chitosan nanoparticles (HACNP/LY) and revealed their anticancer effect and influence on p16-CDK4/6-pRb pathway against colon cell line. The nanoparticle sizes of CNP/LY and HACNP/LY were approximately195±39.6 nm and217±31.1 nm, respectively. The zeta potentials of CNP/LY and HACNP/LY were37.3±1.5 mV and30.3±2.2 mV, respectively. And the preparation process showed considerable drug encapsulation efficiency and loading efficiency. LY2835219, CNP/LY, and HACNP/LY inhibited HT29 cell proliferation with 0.68, 0.54, and 0.30 μM of IC50, respectively. G1 phase was arrested by LY2835219 and its formulations. Furthermore, inhibition of CDK4/6 by LY2835219 formulations induced CDK4, CDK6, cyclin D1, and pRb decrease and p16 increase at both protein and mRNA levels. Overall, nanoparticle formulated LY2835219 could enhance the cytotoxicity and cell cycle arrest, and HACNP/LY strengthened the trend furtherly compared to CNP/LY. It is the first time to demonstrate the anticancer effect and mechanism against HT29 by LY2835219 and its nanoparticles. The drug and its nanoparticle formulations delay the cell growth and arrest cell cycle through p16-CDK4/6-pRb pathway, while the nanoparticle formulated LY2835219 could strengthen the process.

Lactobacillus paracasei subsp. paracasei M5L induces cell cycle arrest and calreticulin translocation via the generation of reactive oxygen species in HT-29 cell apoptosis.

Lactobacillus paracasei subsp. paracasei M5L (L. paracasei M5L) was isolated and co-cultured with HT-29 colon cancer cells to study its anti-colorectal cancer effects and mechanism. Using the MTT assay we found that L. paracasei M5L significantly inhibits HT-29 cell proliferation. Morphological and biochemical characteristics of apoptosis were observed and confirmed by hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM). Lactobacilli could change the cell cycle distribution and induce calreticulin (CRT) translocation from the endoplasmic reticulum to the surface of the cytomembrane. We also determine that vast reactive oxygen species (ROS) were generated, while the activities of superoxide dismutase (SOD) and catalase (CAT) were noticeably diminished following L. paracasei M5L treatment. This study reveals that L. paracasei M5L induces apoptosis in HT-29 cells through ROS generation followed by CRT accompanied endoplasmic reticulum (ER) stress and S phase arrest. These results provide new insights into the possible molecular mechanism of L. paracasei M5L as a novel probiotic with the potential for further application.

Downregulation of CD147 expression by RNA interference inhibits HT29 cell proliferation, invasion and tumorigenicity in vitro and in vivo

We investigated the effect of CD147 silencing on HT29 cell proliferation and invasion. We constructed a novel short hairpin RNA (shRNA) expression vector pYr-mir30-shRNA. The plasmid was transferred to HT29 cells. The expression of CD147, MCT1 (lactate transporters monocarboxylate transporter 1) and MCT4 (lactate transporters monocarboxylate transporter 4) were monitored by quantitative PCR and western blotting, respectively. The MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) activities were determined by gelatin zymography assay, while the intracellular lactate concentration was determined by the lactic acid assay kit. WST-8 assay was used to determine the HT29 cell proliferation and the chemosensitivity. Invasion assay was used to determine the invasion of HT29 cells. In addition, we established a colorectal cancer model, and detected CD147 expression in vivo. The results showed that the expression of CD147 and MCT1 was significantly reduced at both mRNA and protein levels, and also the activity of MMP-2 and MMP-9 was reduced. The proliferation and invasion were decreased, but chemosensitivity to cisplatin was increased. Invivo, the CD147 expression was also significantly decreased, and reduced the tumor growth after CD147 gene silencing. The results demonstrated that silencing of CD147 expression inhibited the proliferation and invasion, suggesting CD147 silencing might be an adjuvant gene therapy strategy to chemotherapy.

Effect of n-3 polyunsaturated fatty acids on proliferation and apoptosis of human colon cancer cell

To investigate the effects of n-3 polyunsaturated fatty acids(n-3PUFA) on human colorectal cancer cell line HT-29 and associated mechanism. The effects of docosahexaenoic acid (DHA) on the proliferation and apoptosis on HT-29 human colorectal cancer cells were evaluated by MTT assay, cell morphology (Hoechst33258 dyeing), DNA gel electrophoresis, and flow cytometry. The content of n-6PUFA and n-3PUFA of the treated cells and the ratio of n-6/n-3PUFA were analyzed by chromatography. DHA effectively inhibited HT-29 cell proliferation in a dose- and time-dependent manner. The proliferative inhibition rates of HT-29 cells treated with 10, 20, 40, and 80 mg/L DHA for 24 hours were 16.8%, 24.7%, 50.0%, and 60.1%, respectively, while the inhibition rates were 50.0%, 69.9%, and 77.0% respectively when HT-29 cells were treated with 40 mg/L DHA for 24, 48, and 72 hours. The typical apoptotic morphologic changes of HT-29 cells could be observed, including chromatin margination, nuclear condensation and apoptotic bodies. Gel electrophoresis of DNA degradation displayed typical DNA ladder fragments. HT-29 cells treated with DHA were arrested in G1 phase and the proportion of HT-29 cells in Gl phase increased compared with that of the control group (72.1% vs. 51.3%) while the proportion of the cells in S phase decreased significantly (19.9% vs. 38.9%). The content of n-6PUFA decreased, n-3PUFA content increased and the ratio of n-6/n-3PUFA lowered significantly in colorectal cancer cells treated with DHA (P<0.01). n-3PUFA can inhibit the growth of human colorectal cancer cells via inhibition of the proliferation and induction of apoptosis. These effects may be associated with decrease in n-6/n-3PUFA ratio.

Phytochemical, Antioxidant, and Antiproliferative Properties of Seed Oil and Flour Extracts of Maryland-Grown Tobacco Cultivars

To determine the possible alternative use of tobacco, the seeds representing seven Maryland tobacco cultivars were investigated for their phytochemical, antioxidant, and antiproliferative properties. Tobacco seed oils were extracted by the Soxhlet method, and analyzed for their yield, density, refractive index, fatty acid profiles, and tocopherol profile. The defatted flours were extracted in 50% acetone and 80% ethanol. The tobacco seed oil and flour extracts were analyzed for total phenolic contents (TPC) and scavenging capacities against peroxyl, hydroxyl and 2,2-diphenyl-1-picryhydrazyl (DPPH) radicals. The fatty acid compositions of phospholipids and the protein content of the flours were also analyzed. In addition, oil and flour extracts of varieties MD609 and MD609LA were evaluated for their antiproliferative effects on HT-29 human colon cancer cells. All of the tested extracts significantly inhibited HT-29 cell proliferation except that from MD609 oil. The data from this study suggest the potential alternative use of tobacco seeds in developing natural antioxidants and antiproliferative agents for improving human health.

A glycoprotein from Laminaria japonica induces apoptosis in HT-29 colon cancer cells

We isolated a novel glycoprotein from the brown alga Laminaria japonica that has antiproliferative effects on HT-29 colon cancer cells. We also identified the mechanism by which this glycoprotein, named LJGP, induces apoptosis. MTS assays showed that LJGP inhibited the proliferation of several cancer cell lines (AGS, HepG2, HT-29) in a dose-dependent manner. Especially in HT-29 cells, proliferation was significantly decreased. LJGP treatment on HT-29 displayed several apoptotic features, such as DNA fragmentation, sub-G1 arrest, caspase-3 activation, and PARP degradation. Consistent with sub-G1 arrest, LJGP decreased the expression of Cdk2, cyclin E, cyclin D1, PCNA, E2F-1, and phosphorylated pRb. Furthermore, the increase of p27 expression was observed. We also determined that LJGP-induced apoptosis leads to the formation of a death-induced signaling complex of Fas, FADD, and procaspase-8. LJGP induced the reduction of mitochondrial membrane potential with activation of the Bcl-2 family of proteins and caspase-9. These findings suggest that LJGP inhibits HT-29 cell proliferation by inducing apoptosis, which may be mediated via multiple pathways, including the Fas signaling pathway, the mitochondrial pathway, and cell cycle arrest. Therefore, LJGP can be a useful treatment option for colon cancer in humans.

Anti-proliferative effects of Lethariella zahlbruckneri extracts in human HT-29 human colon cancer cells

This study was performed to elucidate the anti-proliferative effects and the apoptotic mechanisms of extracts from Lethariella zahlbruckneri in HT-29 human colon cancer cells. Both the acetone extract (AEL) and methanolic extract (MEL) of L. zahlbruckneri decreased viable cell numbers in a dose- and time-dependent manner in HT-29 cells. The AEL showed stronger cytotoxicity than MEL. Cell death induced by AEL increased cell populations in the sub-G1 phase, as well as the formation of apoptotic bodies and nuclear condensation, whereas MEL did not. Therefore, the potential of AEL to induce apoptosis was examined. Apoptosis induced by AEL was associated with the activation of initiator caspases-8 and -9, as well as the effector caspase-3. AEL stimulated Bid cleavage. This indicated that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. AEL increased the expression of the pro-apoptotic protein, Bax, and decreased the expression of the anti-apoptotic protein, Bcl-2. AEL also increased the expression of the caspase-independent mitochondrial apoptosis factor, AIF, in HT-29 cells. These results indicate that AEL inhibited HT-29 cell proliferation by inducing apoptosis, which might be mediated via both caspase-dependent and -independent pathways.

Induction of apoptosis in HT-29 colon cancer cells by crude saponin from Platycodi Radix

This study examined the apoptotic effects of crude saponins acquired from the roots of Platycodon grandiflorum (SPR) in HT-29 human colon cancer cells. SPR decreased HT-29 cell proliferation in dose- and time-dependent manners by inducing apoptosis via DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. The apoptosis induced by SPR was associated with the activation of initiator caspases-8 and -9, as well as the effector caspase-3. SPR stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. SPR increased the expression of the pro-apoptotic protein, Bax, and decreased the expression of the anti-apoptotic protein, Bcl-2. SPR also increased the expression of the caspase-independent mitochondrial apoptosis factor, AIF, in HT-29 cells. These results indicate that SPR inhibits HT-29 cell proliferation by inducing apoptosis, which may be mediated via both caspase-dependent and -independent pathways.