Colon Cancer Cell Death Research Articles

Induction of colon and cervical cancer cell death by cinnamic acid derivatives is mediated through the inhibition of Histone Deacetylases (HDAC).

Recent studies from our group and many others have shown the ability of histone deacetylase (HDAC) inhibitors for retarding the growth of carcinomas of cervix, colon and rectum in vitro. A search for naturally occurring HDAC inhibitors continues due to the adverse effects associated with known HDAC inhibitors like SAHA and TSA. Therefore in the current study, naturally occurring cinnamic acids derivatives were screened for HDAC inhibitory effect using in silico docking method which identified cinnamic acids as potential candidates. Cinnamic acids (CA) are naturally occurring phenolic compounds known to exhibit anticancer properties. However, it is not clearly known whether the anticancer properties of CA derivatives are due to the inhibition of oncogenic HDACs, if so how the efficacy varies among various CA derivatives. Hence, the HDAC inhibitory potential of CA derivatives containing increasing number of hydroxylic groups or methoxy moieties was determined using Discovery Studio software and the most potent CA derivatives tested ex vivo (biochemical assay) as well as in vitro (using cell based assay). Among CA derivatives tested, dihydroxy cinnamic acid (DHCA, commonly known as caffeic acid) exhibited better interactions with HDAC2 (compared to other isoforms) in silico and inhibited its activity ex vivo as well as in vitro. Targeted reduction of HDAC activity using DHCA induced death of cancer cells by (a) generating reactive oxygen species, (b) arresting cells in S and G2/M phases; and (c) induction of caspase-3 mediated apoptosis. In conclusion, we demonstrated that DHCA inhibited cancer cell growth by binding to HDAC followed by the induction of apoptosis.

Ampelopsin-induced reactive oxygen species enhance the apoptosis of colon cancer cells by activating endoplasmic reticulum stress-mediated AMPK/MAPK/XAF1 signaling.

Ampelopsin (Amp) is bioactive natural product and exerts anti-cancer effects against several cancer types. The present study investigated the anti-colon cancer activity of Amp and explored its mechanism of action. The treatment of colon cancer cells with Amp resulted in the dose- and time-dependent induction of apoptosis via the activation of endoplasmic reticulum (ER) stress, 5' adenosine monophosphate-activated protein kinase (AMPK), and c-Jun N-terminal protein kinase (JNK)/p38 mitogen-activated protein kinases (MAPKs). Salubrinal, an ER stress inhibitor, prevented the upregulation of ER stress-associated proteins, including phosphorylated protein kinase RNA-like ER kinase, phosphorylated eukaryotic translation initiation factor 2α, glucose-regulated protein 78, and CCAAT/enhancer-binding protein homologous protein, as well as suppressing AMPK activation and the MAPK signaling pathway. Knockdown of AMPK by RNA interference failed to block ER stress. Additionally, SP600125 (a JNK inhibitor) and SB203580 (a p38-MAPK inhibitor) effectively inhibited apoptosis and attenuated the expression of X-linked IAP-associated factor 1 (XAF1) and apoptotic Bcl-2 family proteins (BCL2 antagonist/killer 1 and BCL2-associated X protein) in Amp-treated colon cancer cells. Furthermore, reactive oxygen species (ROS)-mediated ER stress/AMPK apoptotic signaling pathway in Amp-treated colon cancer cells were markedly inhibited by treatment with N-acetyl-L-cysteine, a ROS scavenger. These results demonstrate that treatment with Amp induces the apoptotic death of colon cancer cells through ER stress-initiated AMPK/MAPK/XAF1 signaling. These results also provide experimental information for developing Amp as therapeutic drug against colon cancer.

Dual Effects of Resveratrol on Cell Death and Proliferation of Colon Cancer Cells

ABSTRACTColorectal cancer remains a main cause of deaths worldwide, and novel agents are being searched to treat this disease. Polyphenols have emerged as promising therapeutic tools in cancer. Resveratrol (3,5,4′-trihydoxy-trans-stilbene) induces cell death in different tumor cell lines, and it also stimulates the proliferation of specific breast and prostate cancer cell lines. Here, we studied the impact of resveratrol over a 100-fold concentration range on cell death and proliferation of HT-29 colorectal adenocarcinoma cells. After 96 h of treatment, a biphasic pattern was observed. At lower concentrations (1 and 10 μmol/l), resveratrol increased the cell number, as did the polyphenol quercetin. At 50 or 100 μmol/l, resveratrol reduced the cell number and increased the percentage of apoptotic or necrotic cells, thus indicating cytotoxicity. On HCT116 colon cancer cells, however, no proliferative properties of resveratrol were observed. Resveratrol-induced cytotoxicity on HT-29 cells was associated with NADPH oxidase activation and increased levels of histone γH2AX, a marker of DNA damage, paralleled by enhanced sirtuin 6 levels, likely as a repair mechanism. Overall, resveratrol may be an effective tool in anti-tumor chemotherapy. However, since under some conditions it may favor tumor cell growth, appropriate local concentrations must be achieved to minimize unwanted effects of resveratrol.

Luteolin induces apoptotic cell death via antioxidant activity in human colon cancer cells.

The present study determined whether luteolin induces HT-29 colon cancer cell death through an antioxidant effect such as the activation of antioxidant enzymes. Luteolin decreased cell viability in human colon cancer cells (HT-29), whereas it had no effect on normal colon cells (FHC). Luteolin induced apoptosis by activating the mitochondria-mediated caspase pathway in HT-29 cells. Luteolin caused loss of the mitochondrial membrane action potential, increased mitochondrial Ca2+ level, upregulated Bax, downregulated Bcl-2, induced the release of cytochromec from mitochondria to the cytosol, and increased the levels of the active forms of caspase-9 and caspase-3. Luteolin-induced apoptosis was accompanied by the activation of intracellular and mitochondrial reactive oxygen species scavenging through the activation of antioxidant enzymes, such as superoxide dismutase and catalase in HT-29 cells. Luteolin increased the level of reduced glutathione (GSH) and the expression of GSH synthetase, which catalyzes the second step of GSH biosynthesis. The apoptotic effect of luteolin was mediated by the activation of the mitogen-activated protein kinase signaling pathway. The present results indicate that luteolin induces apoptosis by promoting antioxidant activity and activating MAPK signaling in human colon cancer cells.

Abstract 1052: Aldose reductase inhibitor increases doxorubicin-sensitivity of colon cancer cells and decreases cardiomyopathy

Abstract Chemotherapy is one of the most common option for colorectal cancer (CRC), specifically at the advanced stages and after surgical resection of the tumor. Anthracycline drugs such as doxorubicin, daunorubicin are the most commonly used for the therapy of leukemia, lymphoma and breast cancer. However, doxorubicin is not a very effective drug to kill CRC cells since a high amount of doxorubicin is required for colon cancer therapy, which leads to unwanted side effects like cardiac toxicity and cardiomyopathy. Therefore, better adjuvant therapies are required to increase sensitivity of CRC to doxorubicin as well as to decrease the associated cardiomyopathy. Our recent studies indicate that aldose reductase (AR; AKR1B1) inhibitors such as fidarestat prevent CRC growth as well as metastasis in nude mice xenograft models. We now examined our hypothesis that AR inhibition increases the sensitivity of CRC to doxorubicin and decreases its cardiotoxicity by modulating oxidative stress-mediated signaling pathways. We have investigated the effect of co-administration of doxorubicin along with AR inhibitor fidarestat in in vitro and in vivo CRC cells growth and toxicity. Our results suggest that treatment of CRC cells with fidarestat increases the efficacy of doxorubicin-induced death of HT-29 and SW-480 colon cancer cells. Flow cytometric analysis indicates that in fidarestat + doxorubicin -treated CRC cells, the intracellular accumulation of doxorubicin is increased as compared to doxorubicin alone-treated cells. Further, AR inhibition prevents doxorubicin-induced increase in the expression of drug transporter proteins such as MRP-1 and ABCG-2 in CRC cells. In addition, combination of fidarestat with doxorubicin significantly prevented the growth of CRC cells in nude mice xenografts as compared to doxorubicin alone. Fidarestat also prevented the doxorubicin-induced cardiac toxicity as measured by serum troponin-I levels and inflammatory cytokines and chemokines in the serum and heart of doxorubicin-treated mice in combination with fidarestat. Most importantly, fidarestat protects the cardiac damage and dysfunction in doxorubicin-treated mice. Thus, our results suggest that fidarestat could be used as a novel adjuvant therapy in enhancing doxorubicin sensitivity of CRC cells and to reduce the doxorubicin-associated cardiotoxicity. Citation Format: Himangshu Sonowal, Pabitra B. Pal, Satish K. Srivastava, Kota V. Ramana. Aldose reductase inhibitor increases doxorubicin-sensitivity of colon cancer cells and decreases cardiomyopathy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1052. doi:10.1158/1538-7445.AM2017-1052

A novel SIRT1 inhibitor, 4bb induces apoptosis in HCT116 human colon carcinoma cells partially by activating p53

The NAD+-dependent protein deacetylase SIRT1 has emerged as an important target for epigenetic therapeutics of colon cancer as its increased expression is associated with cancer progression. Additionally, SIRT1 represses p53 function via deacetylation, promoting tumor growth. Therefore, inhibition of SIRT1 is of great therapeutic interest for the treatment of colon cancer. Here, we report discovery of a novel quinoxaline based small molecule inhibitor of human SIRT1, 4bb, investigated its effect on viability of colon cancer cells and molecular mechanism of action. In vitro, 4bb is a significantly more potent SIRT1 inhibitor, compared to β-naphthols such as sirtinol, cambinol. Increasing concentration of 4bb decrease viability of colon cancer cells but, does not affect the viability of normal dermal fibroblasts depicting cancer cell specificity. Further, 4bb treatment increased p53 acetylation, Bax expression and induced caspase 3 cleavage suggesting that the death of HCT116 colon cancer cells occur through intrinsic pathway of apoptosis. Overall, our results presents 4bb as a new class of human SIRT1 inhibitor and suggest that inhibition of SIRT1 by 4bb induces apoptosis of colon cancer cells at least in part via activating p53 by preventing p53 deacetylation, increasing Bax expression and inducing caspases. Therefore, this molecule provide an opportunity for lead optimization and may help in development of novel, non-toxic epigenetic therapeutics for colon cancer.

Apoptotic Activity of Lactobacillus plantarum DGK-17-Fermented Soybean Seed Extract in Human Colon Cancer Cells via ROS-JNK Signaling Pathway.

Fermented food has been always possesses upper hand compared to normal food due to its antibacterial, antioxidant, and anticancer properties. Soybeans, which have high nutritional value, are widely consumed in Korea. In this study, soybean seed powder fermented with Lactobacillus plantarum DGK-17, which was previously isolated from kimchi, showed anticancer potential. Fermented soybean extract (FSE) resulted in morphological changes, reduction of cancer cell colony formation and apoptotic cell death of HCT-116 colon cancer cells in a dose-dependent manner, and IC50 value of 111 μg. FSE treatment caused reduction of cell growth in a dose-dependent manner via release of lactate dehydrogenase. FSE treatment induced HCT-116 apoptotic cell death as confirmed by the presence of fragmented nuclei, oxidative burst, and reduced mitochondrial membrane potential (ΔΨm ). Further, FSE treatment sensitized cells to ER stress via IRE1-α induction. FSE treatment also resulted in JNK activation, subsequently causing activation of Bax and downregulation of BCl2. Weakened mitochondrial membrane potential (ΔΨm ) also caused release of Cyto C, further activating caspase-mediated cell death. Therefore, this study reveals the apoptotic role of DGK-17-fermented soybean seed extract in human colon cancer HCT-116 cells.

Colorectal cancer cell death by pterostilbene through regulation of Bcl‐2 family signaling pathway

Pterostilbene is natural antioxidant phytochemical from blue berries. We have been demonstrated that pterostilbene stimulates human colon cancer cell death by endoplasmic reticulum (ER) stress, GRP78‐eIFα‐ATF3 pathway, on previous our studies. However, it is still not clear whether pterostilbene regulate Bcl‐2 family signaling pathway undergo apoptosis in colon cancer cells. In the current study, analysis of the gene expression by western blotting and RT‐PCR revealed that pterostilbene modulated the expression of Bcl‐2, Bak1, and Bax in human colon cancer cells. Our results also showed that pterostilbene triggered the expression of scavenger receptor CD36, which is one of the crucial factor cancer cells undergoing mitochondria dependent apoptosis. These observations indicate that pterostilbene as a potent natural chemotherapeutic compound and provide a part of evidence for role of Bcl‐2 family signaling pathway and scavenger receptor CD36 in mediating pterostilbene in human colon cancer cells.

Smad7 knockdown activates protein kinase RNA-associated eIF2\u03b1 pathway leading to colon cancer cell death

Upregulation of Smad7, an inhibitor of transforming growth factor-β1 (TGF-β1), occurs in sporadic colorectal cancer (CRC) and knockdown of Smad7 inhibits CRC cell growth, a phenomenon that associates with decreased expression of cell division cycle 25 homolog A and arrest of cells in the S phase of the cell cycle. These findings occur in CRC cells unresponsive to TGF-β1, thus suggesting the existence of a Smad7-mediated TGF-β1-independent mechanism that controls CRC cell behavior. Here we show that Smad7 inhibition with a specific Smad7 antisense oligonucleotide upregulates eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, a transcription factor involved in the regulation of cell cycle arrest and induction of cell death, and induces activating transcription factor 4 (ATF4) and CCAAT/enhancer binding protein homology protein (CHOP), two downstream targets of eIF2α. Among the upstream kinases that control eIF2α phosphorylation, the serine–threonine protein kinase RNA (PKR), but not general control non-derepressible 2 (GCN2) and protein kinase RNA-like endoplasmic reticulum kinase (PERK), is activated by Smad7 knockdown. PKR silencing abolishes Smad7 antisense-induced eIF2α phosphorylation and ATF4/CHOP induction, thereby preventing Smad7 antisense-driven cell death. Smad7 inhibition diminishes interaction of PKR with protein kinase inhibitor p58 (p58IPK), a cellular inhibitor of PKR, but does not change the expression and/or activity of other factors involved in the control of PKR activation. These findings delineate a novel mechanism by which Smad7 knockdown promotes CRC cell death.

The aryl hydrocarbon receptor as an antitumor target of synthetic curcuminoids in colorectal cancer

BackgroundCurcumin has proven to be a potent antitumor agent in both preclinical and clinical models of colorectal cancer (CRC). It has also been identified as a ligand of the transcription factor known as the aryl hydrocarbon receptor (AHR). Our laboratory has identified the AHR as a mechanism which contributes to both tumorigenesis in a mouse model of inflammatory CRC as well an apoptotic target in vitro. Curcumin's role as an AHR ligand may modulate its effects to induce colon cancer cell death, and this role may be enhanced via structural modification of the curcumin backbone. We sought to determine if the two piperidone analogs of curcumin, RL66 and RL118, exhibit more robust antitumor actions than their parent compound in the context of colorectal cancer in vitro. Moreover, to ascertain the ability of curcumin, RL66 and RL118 to activate the AHR and evaluate if this activation has any effect on CRC cell death. Materials and methodsDLD1, HCT116, LS513, and RKO colon cell lines were propagated in vitro. Natural curcumin was obtained commercially, whereas RL66 and RL118 were synthesized and characterized de novo. Multiwell fluorescent/luminescent signal detection was used to simultaneously ascertain cell viability, cell cytonecrosis, and relative amounts of apoptotic activity. AHR activity was measured with a dual luciferase reporter gene system. Stable expression of small interfering RNA interference was established in the HCT116 cell lines to create AHR “knock down” cell lines. ResultsBoth RL66 and RL118 proved to be more potent antitumor agents than their parent compound curcumin in all cell lines tested. The majority of this cell death was due to induction of apoptosis, which occurred earlier and to a greater degree following RL66 and RL118 treatment as opposed to curcumin. Also, RL66 and RL118 were found to be activators of AHR, and a portion of their ability to cause cell death was dependent on this induction. Curcumin was found unable to activate the AHR, and levels of AHR messenger RNA did not change their effects on cell death. ConclusionsPiperidone analogs of curcumin exhibited enhanced antitumor effects in vitro as opposed to their parent compound. Even more, this enhanced cell death profile may be partially attributed to the ability of these compounds to activate the AHR. Further study of synthetic curcumin analogs as chemopreventives and chemoadjuncts in CRC is warranted. Also, more generally, the AHR may represent a potential putative target for novel anticancer agents for CRC.

5-Hydroxy polymethoxyflavones inhibit glycosaminoglycan biosynthesis in lung and colon cancer cells

5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5HHMF) and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone (5HPMF) are the predominant polymethoxyflavones in orange peel. Here, we showed how 5HHMF or 5HPMF modulated the amount of pre-existing and newly synthesized glycosaminoglycans (GAG) in lung and colon cancer cells. 5HPMF treatment disrupted the formation of FGF/FGFR1c/GAG ternary signaling complex in cell surface. Moreover, 5HHMF remarkably decreased the content of nonsulfated and 3- or 6-sulfated disaccharides of heparan sulfate while no significant changes occurred in that of chondroitin sulfate detected by 1-phenyl-3-methyl-5-pyrazolone (PMP) isotope labeling plus LC/MS analysis. Furthermore, quantification of metabolic incorporating 34S isotope-labeled sulfate into GAG of cultured cells indicated that 5HHMF decreased the sulfated disaccharides of the newly synthesized GAGs by 54%. Overall, the results indicated that 5HHMF and 5HPMF were potential inhibitors of GAG biosynthesis. Our results indicated that GAGs might be the long sought signaling molecules affected by 5HHMF and 5HPMF that lead to lung and colon cancer cell death.

532 - Photodynamic therapy combined with acetylsalicylic acid: the mechanisms involved in colon and esophagus cancer cell death

532 - Photodynamic therapy combined with acetylsalicylic acid: the mechanisms involved in colon and esophagus cancer cell death

LXRβ subcellular localization: A new tool to investigate cancer cell response to LXR ligand-induced cytotoxicity?

Liver X Receptors (LXRs) and their ligands are known for their potential anticancer properties. Recently, our team underlined for the first time that these ligands induce colon cancer cell death through the activation of the inflammasome pathway and in an LXRβ-dependent manner. Moreover, a truncated form of the Retinoid X Receptor α (RXRα), t-RXRα, produced only in cancer cells and not in normal colon epithelial cells interacts with LXRβ to maintain it in the cytoplasm. This specific localization of LXRβ in colon cancer cells dictates their sensitivity towards LXR ligand cytotoxicity whereas its nuclear localization in normal colon epithelial cells prevent it. Our results highlight LXRβ subcellular localization as a promising marker for LXR ligand efficacy in colon cancer treatment.

Inhibition of colon cancer growth by docosahexaenoic acid involves autocrine production of TNFα.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) has anti-inflammatory and anti-cancer properties. Among pro-inflammatory mediators, tumor necrosis factor α (TNFα) plays a paradoxical role in cancer biology with induction of cancer cell death or survival depending on the cellular context. The objective of the study was to evaluate the role of TNFα in DHA-mediated tumor growth inhibition and colon cancer cell death. The treatment of human colorectal cancer cells, HCT-116 and HCT-8 cells, with DHA triggered apoptosis in autocrine TNFα-dependent manner. We demonstrated that DHA-induced increased content of TNFα mRNA occurred through a post-transcriptional regulation via the down-regulation of microRNA-21 (miR-21) expression. Treatment with DHA led to nuclear accumulation of Foxo3a that bounds to the miR-21 promoter triggering its transcriptional repression. Moreover, inhibition of RIP1 kinase and AMP-activated protein kinase α reduced Foxo3a nuclear-cytoplasmic shuttling and subsequent increase of TNFα expression through a decrease of miR-21 expression in DHA-treated colon cancer cells. Finally, we were able to show in HCT-116 xenograft tumor-bearing nude mice that a DHA-enriched diet induced a decrease of human miR-21 expression and an increase of human TNFα mRNA expression limiting tumor growth in a cancer cell-derived TNFα dependent manner. Altogether, the present work highlights a novel mechanism for anti-cancer action of DHA involving colon cancer cell death mediated through autocrine action of TNFα.

The Histone Acetyltransferase GCN5 Expression Is Elevated and Regulated by c-Myc and E2F1 Transcription Factors in Human Colon Cancer.

The histone acetyltransferase GCN5 has been suggested to be involved in promoting cancer cell growth. But its role in human colon cancer development remains unknown. Herein we discovered that GCN5 expression is significantly upregulated in human colon adenocarcinoma tissues. We further demonstrate that GCN5 is upregulated in human colon cancer at the mRNA level. Surprisingly, two transcription factors, the oncogenic c-Myc and the proapoptotic E2F1, are responsible for GCN5 mRNA transcription. Knockdown of c-Myc inhibited colon cancer cell proliferation largely through downregulating GCN5 transcription, which can be fully rescued by the ectopic GCN5 expression. In contrast, E2F1 expression induced human colon cancer cell death, and suppression of GCN5 expression in cells with E2F1 overexpression further facilitated cell apoptosis, suggesting that GCN5 expression is induced by E2F1 as a possible negative feedback in suppressing E2F1-mediated cell apoptosis. In addition, suppression of GCN5 with its specific inhibitor CPTH2 inhibited human colon cancer cell growth. Our studies reveal that GCN5 plays a positive role in human colon cancer development, and its suppression holds a great therapeutic potential in antitumor therapy.

Sphingosine Kinase 2 and Ceramide Transport as Key Targets of the Natural Flavonoid Luteolin to Induce Apoptosis in Colon Cancer Cells.

The plant flavonoid luteolin exhibits different biological effects, including anticancer properties. Little is known on the molecular mechanisms underlying its actions in colorectal cancer (CRC). Here we investigated the effects of luteolin on colon cancer cells, focusing on the balance between ceramide and sphingosine-1-phosphate (S1P), two sphingoid mediators with opposite roles on cell fate. Using cultured cells, we found that physiological concentrations of luteolin induce the elevation of ceramide, followed by apoptotic death of colon cancer cells, but not of differentiated enterocytes. Pulse studies revealed that luteolin inhibits ceramide anabolism to complex sphingolipids. Further experiments led us to demonstrate that luteolin induces an alteration of the endoplasmic reticulum (ER)-Golgi flow of ceramide, pivotal to its metabolic processing to complex sphingolipids. We report that luteolin exerts its action by inhibiting both Akt activation, and sphingosine kinase (SphK) 2, with the consequent reduction of S1P, an Akt stimulator. S1P administration protected colon cancer cells from luteolin-induced apoptosis, most likely by an intracellular, receptor-independent mechanism. Overall this study reveals for the first time that the dietary flavonoid luteolin exerts toxic effects on colon cancer cells by inhibiting both S1P biosynthesis and ceramide traffic, suggesting its dietary introduction/supplementation as a potential strategy to improve existing treatments in CRC.

In vitro cytotoxicity and in vivo efficacy of 5-fluorouracil-loaded enteric-coated PEG-cross-linked chitosan microspheres in colorectal cancer therapy in rats

Purpose: Microspheres of chitosan (CS) cross-linked with polyethylene glycol (PEG) were prepared by emulsion-cross-linking followed by the solvent evaporation technique. The formulations were characterized and subjected to in vitro and in vivo tests to assess cell growth, changes in cell morphology, and activities by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human HT-29 colon cancer cell-lines.Methods: In vivo activity was evaluated for dimethyl hydrazine-induced colorectal cancer in albino male Wistar rats. Biochemical and histological parameters were evaluated to understand their effectiveness for colon cancer therapy.Results: The 5-FU immediate release (IR) formulations suspended in SCMC produced an immediate cytotoxic effect, whereas microspheres inhibited proliferation of tumor cells to induce apoptosis over an extended time. Minimum inhibitory concentration (IC50) values for both standard plain 5-FU and 5-FU-loaded microspheres were respectively 5.00 ± 0.004 µg/mL and 165 ± 1.9 µg/mL which showed the improved safety profile of the microsphere formulation. Tissue distribution showed high concentration of 5-FU in colon that was higher than IC50 value required to stop the growth or death of colon cancer cells from the colonic dysplasia in Duke’s stage A. Significant reduction in tumor volume and multiplicity was observed with increased levels of liver enzymes in animals when treated with standard 5-FU formulation compared with 5-FU loaded microspheres. Elevated levels of serum albumin, creatinine, leukocytopenia, and thrombocytopenia were observed in animals for the standard 5-FU formulation.Conclusion: The PEG cross-linked CS microspheres of this study slowly released 5-FU up to 24 h to colonic region and enhanced the antitumor activity.

In vitro cytotoxicity and in vivo efficacy of 5-fluorouracil-loaded enteric-coated PEG-crosslinked chitosan microspheres in colorectal cancer therapy in rats

Purpose: Microspheres of chitosan (CS) crosslinked with polyethylene glycol (PEG) were prepared by emulsion crosslinking followed by solvent evaporation technique. The formulations were characterized and subjected to in vitro and in vivo tests to assess cell growth, changes in cell morphology and activities by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human HT-29 colon cancer cell lines.Methods: In vivo activity was evaluated for dimethyl hydrazine-induced colorectal cancer in albino male Wistar rats. Biochemical and histological parameters were evaluated to understand their effectiveness for colon cancer therapy.Results: The 5-FU immediate release (IR) formulations suspended in sodium carboxymethyl cellulose (SCMC) produced an immediate cytotoxic effect, whereas microspheres inhibited the proliferation of tumor cells to induce apoptosis over an extended time. Minimum inhibitory concentration (IC50) values for both standard plain 5-FU and 5-FU-loaded microspheres were, respectively, 5.00 ± 0.004 µg/mL and 165 ± 1.9 µg/mL which showed the improved safety profile of the microsphere formulation. Tissue distribution showed high concentration of 5-FU in colon that was higher than IC50 required to stop the growth or death of colon cancer cells from the colonic dysplasia in Duke’s Stage A. Significant reduction in tumor volume and multiplicity was observed with increased levels of liver enzymes in animals when treated with standard 5-FU formulation compared to 5-FU-loaded microspheres. Elevated levels of serum albumin, creatinine, leukocytopenia and thrombocytopenia were observed in animals for the standard 5-FU formulation.Conclusion: The PEG-crosslinked CS microspheres of this study slowly released 5-FU up to 24 h to colonic region and enhanced the antitumor activity.

Shogaol overcomes TRAIL resistance in colon cancer cells via inhibiting of survivin.

In this study, we showed the ability of representative shogaol, which as a major component of ginger, to overcome TRAIL resistance by increasing apoptosis in colon cancer cells. Shogaol increased death receptor 5 (DR5) levels. Furthermore, shogaol decreased the expression level of antiapoptotic proteins (survivin and Bcl-2) and increased pro-apoptotic protein, Bax. Shogaol treatment induced apoptosis and a robust reduction in the levels of the antiapoptotic protein survivin but did not affect the levels of many other apoptosis regulators. Moreover, knockdown of survivin sensitized colon cancer cells to resistant of TRAIL-induced apoptosis. Therefore, we showed the functions of shogaol as a sensitizing agent to induce cell death of TRAIL-resistant colon cancer cells. This study gives rise to the possibility of applying shogaol as an antitumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant colon tumor therapy.

Mechanisms underlying 3-bromopyruvate-induced cell death in colon cancer.

3-Bromopyruvate (3BP) is an energy-depleting drug that inhibits Hexokinase II activity by alkylation during glycolysis, thereby suppressing the production of ATP and inducing cell death. As such, 3BP can potentially serve as an anti-tumorigenic agent. Our previous research showed that 3BP can induce apoptosis via AKT /protein Kinase B signaling in breast cancer cells. Here we found that 3BP can also induce colon cancer cell death by necroptosis and apoptosis at the same time and concentration in the SW480 and HT29 cell lines; in the latter, autophagy was also found to be a mechanism of cell death. In HT29 cells, combined treatment with 3BP and the autophagy inhibitor 3-methyladenine (3-MA) exacerbated cell death, while viability in 3BP-treated cells was enhanced by concomitant treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk) and the necroptosis inhibitor necrostatin (Nec)-1. Moreover, 3BP inhibited tumor growth in a SW480 xenograft mouse model. These results indicate that 3BP can suppress tumor growth and induce cell death by multiple mechanisms at the same time and concentration in different types of colon cancer cell by depleting cellular energy stores.