Oroxylin A improves the sensitivity of HT-29 human colon cancer cells to 5-FU through modulation of the COX-2 signaling pathway

5-Fluorouracil (5-FU) is a principal drug for the treatment of colorectal cancer. Due to its low response and high toxicity, synergistic effects of 5-FU in combination with other drugs have been widely researched. This study investigated whether oroxylin A improved the sensitivity of HT-29 human colon cancer cells to 5-FU. A correlation between COX-2 inhibition by oroxylin A and a synergistic effect of 5-FU on the growth of HT-29 cells was observed, and a COX-2 pathway for this effect was recognized; oroxylin A evidently elevated the level of reactive oxygen species in HT-29 cells, which subsequently inhibited COX-2 expression and enhanced the susceptibility of HT-29 cells to 5-FU. Likely also related to COX-2 inhibition, oroxylin A decreased PGE(2) levels in HT-29 cells. The synergistic effect of 5-FU induced by oroxylin A was also found in the suppression of Bcl-2 and in the activation of P53, Bax, PARP, and procaspase-3 proteins in HT-29 cells. Ultimately, a combination of 5-FU with oroxylin A significantly reduced the growth of HT-29 tumors in nude mice compared with treatment with 5-FU or oroxylin A alone. In conclusion, a combination of 5-FU and oroxylin A has a significant synergistic effect in the inhibition of HT-29 cell proliferation in vitro and controls HT-29 tumor growth in vivo. This synergistic effect may be mainly related to COX-2 inhibition by oroxylin A in HT-29 cells.     

Over-expression of Bcl-2 against Pteris semipinnata L-induced apoptosis of human colon cancer cells via a NF-kappa B-related pathway

Ent-11 alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F), an antitumor component, is a chemical compound isolated from Pteris semipinnata L (PsL), a Chinese traditional herb. We examined whether 5F could affect apoptosis in human colon cancer HT-29 cells, and test whether and how the over-expression of Bcl-2 and Bcl-xL could offset the effect of 5F on cell growth. The result demonstrated that 5F significantly induced apoptosis of HT-29, as shown by MTT assay and DNA fragmentation measurement. Treatment of HT-29 with 5F increased both p38 and iNOS levels, suggesting these two molecules may contribute to the apoptotic effect of 5F. Over-expression of Bcl-2 or Bcl-xL attenuated the increase of p38 and iNOS induced by 5F. The cells with Bcl-2 or Bcl-xL over-expression showed an elevation of nuclear factor kappa B (NF-kappa B) activity, accompanying a significant reduction of 5F-induced apoptosis. Furthermore, inhibition of NF-kappa B by I k B alpha SR, which is a powerful inhibitor of NF-kappa B, restored the ability of 5F to induce apoptosis in the cells transfected with Bcl-2. These data strongly indicated that the apoptotic effect of 5F on HT-29 was closely associated with the activity of NF-kappa B, which was up-regulated by Bcl-2 and Bcl-xL. In conclusion, 5F induced apoptosis in HT-29 cells and this apoptotic effect was associated with the high level of p38 and iNOS expression. The apoptotic effect of 5F could be significantly offset by over-expression of either Bcl-2 or Bcl-xL. Bcl-2, and to the less extent, Bcl-xL, were able to increase the activity of NF-kappa B, which was a known anti-apoptotic molecule in human colon cancer cells.     

Quercetin decreases the expression of ErbB2 and ErbB3 proteins in HT-29 human colon cancer cells

Quercetin has chemoprotective properties in experimental colon cancer models, and in vitro studies have demonstrated that quercetin inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 receptor tyrosine kinases have been associated with the development of human colon cancer, and the expressions of both receptors are high in HT-29 cells. In this study, we assessed quercetin regulation of HT-29 and SW480 cell apoptosis and the influence of quercetin on the protein expression of ErbB2, ErbB3, Akt, Bax and Bcl-2. We cultured HT-29 cells in the presence of various concentrations (0, 25, 50, or 100 micromol/L) of quercetin or rutin. Quercetin inhibited HT-29 cell growth in a dose-dependent manner, whereas rutin had no effect on the cell growth. DNA that was isolated from cells treated with 50 micromol/L of quercetin exhibited an oliogonucleosomal laddering pattern characteristic of apoptotic cell death. Western blot analysis of cell lysates revealed that Bcl-2 levels decreased dose-dependently in cells treated with quercetin, but Bax remained unchanged. Quercetin increased levels of cleaved caspase-3 and the 89-kDa fragment of poly (ADP-ribose) polymerase. In addition, phosphorylated Akt levels were markedly lower in cells treated with 25 micromol/L quercetin, but total Akt levels decreased only at 100 micromol/L quercetin. Furthermore, a dose-dependent decrease in ErbB2 and ErbB3 levels was detected in quercetin-treated cells. The results obtained using SW480 cells were similar to those obtained with HT-29 cells. In conclusion, we have shown that quercetin inhibits cell growth and induces apoptosis in colon cancer cells, and that this may be mediated by its ability to down-regulate ErbB2/ErbB3 signaling and the Akt pathway.     

Effect of lycopene as an adjuvant therapy with 5-florouracil in human colon cancer

Colon cancer (CC) is among the most frequent human cancers. Although, there is improvement in diagnostic techniques and existing treatment possibilities. Still, there is an unmet need for a novel treatment regimen that will improve the patient's quality of life. Here, the role of lycopene as an adjuvant therapy with 5-fluorouracil (5-FU) was explored in Caco2 colon cancer cells. Cells were exposed to a dose (3 µg/ml) of 5-FU and three doses (60, 90, 120 µg/ml) of lycopene either alone or as a mixture with 5-FU. Cytotoxicity, genotoxicity, oxidative stress, gene expression, and apoptotic parameters were investigated in this study. Findings showed that 5-FU or lycopene alone induced a dose-dependent increase in cytotoxicity which was slightly reduced in lycopene mixtures. Apoptotic assays showed that 5-FU induced a significant level of apoptosis but not necrosis. However, a lycopene mixture with 5-FU enhanced 5-FU triggered apoptosis and promoted necrosis. The mixtures were also shown to suppress mitochondrial membrane potential while gene expression analyses showed the induction of Bax expression upon exposure to mix 90 exhibited the highest Bax to Bcl-2 ratio and caspase 3 and 9 gene expression. Furthermore, the mixture treatment also inhibited cell migration in the wound healing assay compared to 5-FU alone. In conclusion, lycopene was found to sensitize Caco 2 cell lines to 5-FU treatment by inducing the expression of apoptotic genes. This, coupled with lycopene suppression of cytotoxicity and cell migration, indicates lycopene may be a promising candidate for adjuvant therapy involving 5-FU in CC.     

Addition of anticancer agents enhances freezing-induced prostate cancer cell death: implications of mitochondrial involvement

Recent evidence suggests that the successful treatment of prostate cancer may require adjuvant therapies. Accordingly, a better understanding of the molecular mechanisms involved in current treatments may lead to enhanced efficacy by providing a basis for adjuvant therapies. In this study, we demonstrate that the combination of sub-lethal concentrations of chemotherapeutic agents prior to freezing (-15 degrees C) in a prostate cancer cell (PC-3) model results in enhanced efficacy over either treatment alone. Morphological analysis revealed that necrosis appeared to be the prevalent mode of cell death following adjuvant (in vitro) modeling, yet molecular analysis indicated that freezing and chemotherapy differentially activated apoptotic cascades through modulating opposing members of the Bcl-2 protein family. Freezing results in a time-dependent increase of the antiapoptotic Bcl-2 protein, while chemotherapy results in an increase of the pro-apoptotic Bax protein. Anti-apoptotic Bcl-2 protein levels increase over 3-fold following exposure to freezing. 5-Fluorouracil (5-FU) causes pro-apoptotic Bax levels to increase 2-fold during the drug exposure. The increase in Bax was also apparent following the combination of 5-FU/freezing, while Bcl-2 levels were maintained at or below control levels. This led to a shift in the Bcl-2 to Bax ratio to a pro-death tendency. Other effective cryo/chemo combinations were also found to provide similar effects. The combination of cisplatin/freezing resulted in a 4-fold increase in the ratio of Bax to Bcl-2 when compared to controls, which represented a 2-fold increase over the 5-FU/freezing-combination model. This increase may contribute to the continued reduction in cell number observed during the 13-day recovery period. Additionally, the addition of an apoptotic caspase inhibitor was not able to protect cultures from cell death following combination treatment. In conclusion, the data suggest that both Bcl-2 and Bax may, not only, play an important role in the efficacy of the cryo/chemo combination, but also the balance between the two may determine the role and extent of system destruction.     

Combination of 5-fluorouracil and genistein induces apoptosis synergistically in chemo-resistant cancer cells through the modulation of AMPK and COX-2 signaling pathways

5-Fluorouracil (5-FU) is one of the widely used chemotherapeutic drugs targeting various cancers, but its chemo-resistance remains as a major obstacle in clinical settings. In the present study, HT-29 colon cancer cells were markedly sensitized to apoptosis by both 5-FU and genistein compared to the 5-FU treatment alone. There is an emerging evidence that genistein, soy-derived phytoestrogen, may have potential as a chemotherapeutic agent capable of inducing apoptosis or suppressing tumor promoting proteins such as cyclooxygenase-2 (COX-2). However, the precise mechanism of cellular cytotoxicity of genistein is not known. The present study focused on the correlation of AMPK and COX-2 in combined cytotoxicity of 5-FU and genistein, since AMPK is known as a primary cellular homeostasis regulator and a possible target molecule of cancer treatment, and COX-2 as cell proliferation and anti-apoptotic molecule. Our results demonstrated that the combination of 5-FU and genistein abolished the up-regulated state of COX-2 and prostaglandin secretion caused by 5-FU treatment in HT-29 colon cancer cells. These appear to be followed by the specific activation of AMPK and the up-regulation of p53, p21, and Bax by genistein. Under same conditions, the induction of Glut-1 by 5-FU was diminished by the combination treatment with 5-FU and genistein. Furthermore, the reactive oxygen species (ROS) was found as an upstream signal for AMPK activation by genistein. These results suggested that the combination of 5-FU and genistein exert a novel chemotherapeutic effect in colon cancers, and AMPK may be a novel regulatory molecule of COX-2 expression, further implying its involvement in cytotoxicity caused by genistein.     

Targeting miR-21 enhances the sensitivity of human colon cancer HT-29 cells to chemoradiotherapy in vitro

5-Fluorouracil (5-FU) is a classic chemotherapeutic drug that has been widely used for colorectal cancer treatment, but colorectal cancer cells are often resistant to primary or acquired 5-FU therapy. Several studies have shown that miR-21 is significantly elevated in colorectal cancer. This suggests that this miRNA might play a role in this resistance. In this study, we investigated this possibility and the possible mechanism underlying this role. We showed that forced expression of miR-21 significantly inhibited apoptosis, enhanced cell proliferation, invasion, and colony formation ability, promoted G1/S cell cycle transition and increased the resistance of tumor cells to 5-FU and X radiation in HT-29 colon cancer cells. Furthermore, knockdown of miR-21 reversed these effects on HT-29 cells and increased the sensitivity of HT-29/5-FU to 5-FU chemotherapy. Finally, we showed that miR-21 targeted the human mutS homolog2 (hMSH2), and indirectly regulated the expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). These results demonstrate that miR-21 may play an important role in the 5-FU resistance of colon cancer cells.     

Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29

Autophagy is a major catabolic process allowing the renewal of intracellular organelles by which cells maintain their homeostasis. We have previously shown that autophagy is controlled by two transduction pathways mediated by a heterotrimeric Gi3 protein and phosphatidylinositol 3-kinase activities in the human colon cancer cell line HT-29. Here, we show that 3-methyladenine, an inhibitor of autophagy, increases the sensitivity of HT-29 cells to apoptosis induced by sulindac sulfide, a nonsteroidal anti-inflammatory drug which inhibits the cyclooxygenases. Similarly, HT-29 cells overexpressing a GTPase-deficient mutant of the G(alpha i3) protein (Q204L), which have a low rate of autophagy, were more sensitive to sulindac sulfide-induced apoptosis than parental HT-29 cells. In both cell populations we did not observe differences in the expression patterns of COX-2, Bcl-2, Bcl(XL), Bax, and Akt/PKB activity. However, the rate of cytochrome c release was higher in Q204L-overexpressing cells than in HT-29 cells. These results suggest that autophagy could retard apoptosis in colon cancer cells by sequestering mitochondrial death-promoting factors such as cytochrome c.     

INrf2 (Keap1) targets Bcl-2 degradation and controls cellular apoptosis

Cytosolic inhibitor of Nrf2 (INrf2) is an adaptor protein that mediates ubiquitination/degradation of NF-E2-related factor 2 (Nrf2), a master regulator of cytoprotective gene expression. In this paper, we demonstrate that INrf2 degrades endogenous antiapoptotic B-cell CLL/lymphoma 2 (Bcl-2) protein and controls cellular apoptosis. The DGR domain of INrf2 interacts with the BH2 domain of Bcl-2 and facilitates INrf2:Cul3–Rbx1-mediated ubiquitination of Bcl-2 by the conjugation of ubiquitin molecules to lysine17 of Bcl-2. Further studies showed that INrf2 enhanced etoposide-mediated accumulation of Bax, increased release of cytochrome c from mitochondria, activated caspase-3/7, and enhanced DNA fragmentation and apoptosis. Antioxidants antagonized Bcl-2:INrf2 interaction, led to the release and stabilization of Bcl-2, increased Bcl-2:Bax heterodimers and reduced apoptosis. Moreover, dysfunctional/mutant INrf2 in human lung cancer cells failed to degrade Bcl-2, resulting in decreased etoposide and UV/γ radiation-mediated DNA fragmentation. These data provide the first evidence of INrf2 control of Bcl-2 and apoptotic cell death, with implications in antioxidant protection, survival of cancer cells containing dysfunctional INrf2, and drug resistance.     

去乙酰化酶抑制剂TSA介导Ku70乙酰化对结肠癌HT29细胞凋亡和自噬的影响

目的:探讨不同浓度组蛋白去乙酰化酶抑制剂TSA对结肠癌HT29细胞的增殖、凋亡和自噬影响及其机制研究。方法:取对数生长期人结肠癌HT29细胞,采用MTT法检测不同浓度TSA处理对其细胞活力影响,并根据IC50值确定适宜给药浓度;采用流式细胞术检测不同浓度TSA处理后结肠癌HT29细胞的凋亡情况;Western blot验证空白对照组与TSA给药处理组中凋亡标志蛋白Ku70、acetrl-Ku70、Caspase3、Bax、Bcl-2和自噬标志蛋白LC3和Beclin1的表达。结果:MTT法实验结果表明TSA对结肠癌HT29细胞具有时间和浓度依赖性抑制作用,根据IC50=1.12μM,本研究中TSA的给药浓度为0.5μM和1μM;流式细胞凋亡检测结果表明TSA能够显著促进结肠癌HT29细胞凋亡,且其促凋亡作用存在浓度依赖性;此外,Western blot检测结果证实,与空白对照组相比,TSA给药处理可显著上调上述细胞中acetrl-Ku70以及促凋亡蛋白Caspase3、Bax和自噬标志蛋白LC3和Beclin1的表达,下调抗凋亡蛋白Bcl-2的表达(P<0.05)。结论:组蛋白去乙酰化酶抑制剂(TSA)的体外抗结肠癌细胞的增殖、促进细胞凋亡和自噬作用与其上调Ku70蛋白乙酰化密切相关,有望成为临床潜在抗癌靶点。     

Differential mucin expression in colon carcinoma HT-29 clones with variable resistance to 5-fluorouracil and methotrexate

A current challenge is to define the biological characteristics of colon tumor cells resistant to chemotherapy. Distinct sub-populations of mucus-secreting cells were previously obtained from the colon cancer cell line HT-29 after long-term treatment with the anti-cancer drugs, 5-fluorouracil (5-FU) and methotrexate (MTX). Since mucins are increasingly implicated as playing a role in carcinogenesis, we studied the pattern of mucin expression in two HT-29 clones of mucus-secreting and two clones of enterocyte-like phenotype which differ in their capacity to resist to 5-FU and/or MTX. The expression of both transmembrane (MUC1, MUC3, MUC4) and secreted gel-forming (MUC2, MUC5AC, MUC5B, MUC6) mucins in clones was studied by northern and/or western blotting. The four HT-29 clones showed three cellular phenotypes: (1) The mucus-secreting clone HT29-5F12 consists of unpolarized cells with mucus secretions that have anti-colonic mucin immunoreactivity, and mainly expresses MUC2 and is resistant to 5-FU and sensitive to MTX; (2) The mucus-secreting clone HT29-5M21 forms a monolayer of polarized cells with strong anti-gastric mucin immunoreactivity and mainly expresses MUC5AC and MUC5B and is resistant to MTX and sensitive to 5-FU; (3) The two enterocyte-like clones, HT29-5F7 and HT29-5M12 are resistant to both MTX and 5-FU and express mainly MUC1 and MUC5B, respectively. These clones which originate from a same colorectal tumour and display different patterns of mucin expression as well as differing resistance to MTX and 5-FU will make useful in vitro models for studying the potential role of mucins or other biological markers in drug resistance pathways.     

双歧杆菌脂磷壁酸与5-氟尿嘧啶体内联用对肿瘤细胞凋亡的影响

目的研究双歧杆菌脂磷壁酸与5-氟尿嘧啶联用诱导H22荷瘤小鼠肿瘤细胞凋亡的作用机制。方法双歧杆菌脂磷壁酸联合5-FU处理H22荷瘤Balb/c小鼠,计算抑瘤率,观察小鼠生存期;采用Real time-PCR和Western blot方法分别检测荷瘤小鼠肿瘤组织Bcl-2、Bax和Caspase-3 mRNA及蛋白的表达变化。结果双歧杆菌脂磷壁酸联合5-FU应用,与单独5-FU处理组比较,不仅抑瘤率明显提高（P〈0.01）,且荷瘤小鼠存活时间明显延长（P〈0.01）,肿瘤组织Bcl-2表达下降,Bax和Caspase-3表达升高（P〈0.05）。结论双歧杆菌脂磷壁酸联合5-FU可通过上调Bax和Caspase-3,下调Bcl-2,促进肿瘤细胞凋亡,从而发挥协同抗肿瘤作用。     

Antitumour activity on extrinsic apoptotic targets of the triterpenoid maslinic acid in p53-deficient Caco-2 adenocarcinoma cells

We report that a novel triterpenoid, (2a,3b)-2,3-dihydroxyolean-12-en-28-oic acid (maslinic acid), isolated from olive pomace from Olea europaea, triggers primarily the extrinsic and later the intrinsic apoptotic pathways in Caco-2 human colon-cancer cells. Apoptosis induced by maslinic acid was confirmed by FACS analysis using annexine-V FICT staining. This induction of apoptosis was correlated with the early activation of caspase-8 and caspase-3, the activation of caspase-8 was also correlated with higher levels of Bid cleavage and decreased Bcl-2, but with no change in Bax expression. Maslinic acid also induced a sustained activation of c-Jun N-terminal kinase (JNK). Incubation with maslinic acid also resulted in the later activation of caspase-9, which, together with the lack of any Bax activation, suggests that the mitochondrial pathway is not required for apoptosis induced by maslinic acid in this cell line. In this study we found that the mechanism of apoptotic activation in p53-deficient Caco-2 cells differs significantly from that found in HT-29 cells. Natural agents able to activate both the extrinsic and intrinsic apoptotic pathways by avoiding the mitochondrial resistance mechanisms may be useful for treatment against colon cancer regardless of its aetiology.     

Cyclotrisiloxan and β-Sitosterol rich Cassia alata (L.) flower inhibit HT-115 human colon cancer cell growth via mitochondrial dependent apoptotic stimulation

Cancer traits dependent chemo and radiotherapy display acute toxicity and long-term side effects. Since last two decades, researchers investigated a new anticancer agents derived from plants. Cassia alata (L.) is a medicinal herb distributed in the tropical and humid regions. In this study, C. alata flower methanol extract (CME) have been prepared using cold percolation method and the phytochemical components were identified using GC–MS analysis. CME have been used to study the antiproliferative and apoptosis properties against human colon cancer HT-115 colon cancer cells, its molecular mechanism have been explored. 0.2 mg/mL dose of CME, inhibited 50% of HT-115 colon cancer cell growth after 48hr was confirmed the significant antiproliferation effect. In normal cells such as Vero cells and hMSCs, 0.2 mg/mL dose of CME shown only 4% and 5% growth inhibition confirmed the HT-115 cell specific cytotoxic effect. This effect might be due to the availability of phytoactive biomolecules in CME such as, cyclotrisiloxan, beta-sitosterol and alpha-tocopherol have been confirmed by GC–MS. Most interestingly, PI and AO/ErBr staining of CME treated HT-115 cells shown early (25%), pro (17%) and late (8%) apoptotic and 3% necrotic cells after 48 hr. Treatment with CME extract showed potential effect on the inhibition of protumorigenic inflammatory and oxidative stress genes. Protumorigenic COX-2/PGE-2 and TNF-α/NF-κB immune axis were normalized after CME treatment. Amounts of both apoptosis related mRNA p53, Bax, caspase 3 and p21 genes were upregulated, whereas it resulted in significant reduction in the anti-apoptotic marker mdm2 and Bcl-2 genes. In conclusion, bioactive compounds present in CME potentially inhibit HT-115 colon cancer cell proliferation via an inhibition of protumorigenic immune axis and stimulation of mitochondria dependent apoptotic pathway without necrotic effect.     

Cell cycle arrest and apoptosis induced by methyl 3,5-dicaffeoyl quinate in human colon cancer cells: Involvement of the PI3K/Akt and MAP kinase pathways

Methyl 3,5-dicaffeoyl quinate (MDQ) is a flavonoid glucoside found in several plants that scavenges 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and peroxynitrite, and inhibits the formation of cholesteryl ester hydroperoxide during the copper ion-induced oxidation of blood plasma in rats. In this study, MDQ inhibited proliferation and induced apoptosis in HT-29 cells in a dose-dependent manner as detected by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), trypan blue exclusion, and flow cytometric assays. Western blot analysis showed that apoptosis was dependent on caspase-3 activity. PARP cleavage and the cytosolic release of cytochrome c from mitochondria increased significantly. In addition, these events were accompanied by a collapse in the mitochondrial membrane potential and a decreased Bcl-2/Bax ratio. Furthermore, the MDQ-induced G₀/G₁ arrest was correlated with an increase in p27 and a decrease in cyclin D1 and p53. MDQ also inhibited the phosphorylation of PI3K/Akt and ERK; significantly reduced NF-κB; and in general displayed a significant anti-proliferative effect via a cell cycle arrest and apoptotic induction in HT-29 cells. These results suggest that MDQ has therapeutic potential against human colon carcinoma.     

GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10μM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (?Ψm). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21Waf1/Cip1 was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p     

Subchronic methionine load induces oxidative stress and provokes biochemical and histological changes in the rat liver tissue

Melatonin has antitumor activity via several mechanisms including its anti-proliferative and pro-apoptotic effects. Moreover, it has been proven that melatonin in combination with chemotherapeutic agents enhances chemotherapy-triggered apoptosis in several types of cancer. Therefore, this study was intended to evaluate whether melatonin is able to strengthen the anti-cancer potential of different chemotherapeutic drugs in human colorectal adenocarcinoma HT–29 cells. We found that treatment with 20 µM cisplatin (CIS) or 1 mM 5-fluorouracil (5-FU) for 72 h induced a decrease in HT-29 cell viability. Furthermore, 1 mM melatonin significantly (P < 0.05) increased the cytotoxic effects of 5-FU. Likewise, simultaneous stimulation with 1 mM melatonin and 1 mM 5-FU significantly (P < 0.05) enhanced the ratio of cells with an overproduction of intracellular reactive oxygen species and substantially augmented the population of apoptotic cells compared to the treatment with 5-FU alone. Nonetheless, melatonin only displayed moderate chemosensitizing effects in CIS-treated HT-29 cells, as suggested by a slight increment in the fraction of early apoptotic cells that was observed only after 48 h. Consistently, co-stimulation of HT-29 cells with 20 µM CIS or 1 mM 5-FU in the presence of 1 mM melatonin further increased caspase-3 activation. Apart from this, the cytostatic activity displayed by CIS due to S phase arrest was not affected by concomitant stimulation with melatonin. Overall, our results indicate that melatonin increases the sensitivity of HT-29 cells to 5-FU treatment and, consequently, the indolamine could be potentially applied to colorectal adenocarcinoma treatment as a potent chemosensitizing agent.     

Cyclotrisiloxan and β-Sitosterol rich Cassia alata (L.) flower inhibit HT-115 human colon cancer cell growth via mitochondrial dependent apoptotic stimulation

Cancer traits dependent chemo and radiotherapy display acute toxicity and long-term side effects. Since last two decades, researchers investigated a new anticancer agents derived from plants. Cassia alata (L.) is a medicinal herb distributed in the tropical and humid regions. In this study, C. alata flower methanol extract (CME) have been prepared using cold percolation method and the phytochemical components were identified using GC–MS analysis. CME have been used to study the antiproliferative and apoptosis properties against human colon cancer HT-115 colon cancer cells, its molecular mechanism have been explored. 0.2 mg/mL dose of CME, inhibited 50% of HT-115 colon cancer cell growth after 48hr was confirmed the significant antiproliferation effect. In normal cells such as Vero cells and hMSCs, 0.2 mg/mL dose of CME shown only 4% and 5% growth inhibition confirmed the HT-115 cell specific cytotoxic effect. This effect might be due to the availability of phytoactive biomolecules in CME such as, cyclotrisiloxan, beta-sitosterol and alpha-tocopherol have been confirmed by GC–MS. Most interestingly, PI and AO/ErBr staining of CME treated HT-115 cells shown early (25%), pro (17%) and late (8%) apoptotic and 3% necrotic cells after 48 hr. Treatment with CME extract showed potential effect on the inhibition of protumorigenic inflammatory and oxidative stress genes. Protumorigenic COX-2/PGE-2 and TNF-α/NF-κB immune axis were normalized after CME treatment. Amounts of both apoptosis related mRNA p53, Bax, caspase 3 and p21 genes were upregulated, whereas it resulted in significant reduction in the anti-apoptotic marker mdm2 and Bcl-2 genes. In conclusion, bioactive compounds present in CME potentially inhibit HT-115 colon cancer cell proliferation via an inhibition of protumorigenic immune axis and stimulation of mitochondria dependent apoptotic pathway without necrotic effect.     

Concomitant supplementation of lycopene and eicosapentaenoic acid inhibits the proliferation of human colon cancer cells

Several studies indicated that people who live in the Mediterranean region have very low rates of chronic diseases such as cardiovascular disease and cancer. It is well known that Mediterranean-style diet is rich in vegetables, tomato, fruit, fish and olive oil. These important dietary components may contribute to lower risk of cancer. Lycopene, a major component in tomato, exhibited potential anticarcinogenic activity. Previous studies showed that consumption of fish containing eicosapentaenoic acid (EPA) correlated with reduced risk of cancer. However, the combined effects of lycopene and EPA on the proliferation of human colon cancer have not been studied well yet. Thus, we investigated the anticancer properties and therapeutic potential of lycopene and EPA in human colon cancer HT-29 cells. In this study, we determined the combined effects of lycopene and EPA on the proliferation of human colon cancer HT-29 cells. We demonstrated that low concentration of lycopene and EPA could synergistically inhibit the proliferation of colon cancer cells. The inhibitory mechanism was associated with suppression of phosphatidylinositol 3-kinase/Akt signaling pathway. Furthermore, treatment of lycopene and EPA also synergistically blocked the activation of downstream mTOR molecule. Immunocytochemical staining results revealed that lycopene and EPA could also up-regulate the expression of apoptotic proteins such as Bax and Fas ligand to suppress cell survival. In conclusion, our novel findings suggest that lycopene and EPA synergistically inhibited the growth of human colon cancer HT-29 cells even at low concentration. The inhibitory effects of lycopene and EPA on cell proliferation of human colon cancer HT-29 cells were, in part, associated with the down-regulation of the PI-3K/Akt/mTOR signaling pathway.