The phosphanegold(I) carbonimidothioates, Ph3PAu{SC(OR)=NC6H4Me-4} for R = Me (1), Et (2) and iPr (3), feature linear P-Au-S coordination geometries and exhibit potent in vitro cytotoxicity against HT-29 colon cancer cells in both monolayer and multi-cellular spheroid models (e.g., IC50 = 11.9 ± 0.4 and 20.3 ± 0.3 μM for 2, respectively). Both intrinsic and extrinsic pathways of apoptosis are demonstrated by human apoptosis PCR array analysis, caspase activities, DNA fragmentation and cell apoptotic assays. Compounds 1-3 induce an extrinsic pathway that leads to down-regulation of NFκB. Compound 2 also exhibits an extrinsic apoptotic pathway involving the activation of both p53 and p73, whereas 3 activates p53 only. Lys48- and Lys63-linked polyubiquitination are also promoted by 1-3. Each of cytotoxic Ph3PAu{SC(OR)=NC6H4Me-4}, for R = Me (1), Et (2) and iPr (3), induce an intrinsic apoptotic pathway as well as an extrinsic pathway leading to down-regulation of NFκB. Lys48- and Lys63-linked polyubiquitination are promoted by 1-3 and these are able to inhibit cell invasion and to suppress the activity of TrxR.
Chalcones and their derivatives belong to the flavonoid family. They have been extensively studied for their anticancer properties and some have been approved for clinical use. In this study, the in vivo anti-tumor activity of flavokawain C (FKC), a naturally occurring chalcone found in Kava (Piper methysticum Forst) was evaluated in HCT 116 cells (colon carcinoma). We also attempted to identify potential biomarkers and/or molecular targets in serum with applicability in predicting treatment outcome. The anti-tumor effects and toxicity of FKC were assessed using the xenograft nude mice model. Cisplatin was used as positive control. The anti-proliferative and apoptotic activities were then evaluated in tumor tissues treated with FKC. Furthermore, two-dimensional electrophoresis (2-DE) followed by protein identification using MALDI-TOF/TOF-MS/MS was performed to compare the serum proteome profiles between healthy nude mice and nude mice bearing HCT 116 tumor treated with vehicle solution and FKC, respectively. Our results showed that FKC treatment significantly inhibited HCT 116 tumor growth. In vivo toxicity studies showed that administration of FKC did not cause damage to major organs and had no significant effect on body weight. FKC was found to induce apoptosis in tumor, and this was associated with increased expression of cleaved caspase-3 and decreased expression of Ki67 in tumor tissues. Our proteomic analysis identified five proteins that changed in abundance - Ig mu chain C region (secreted form), GRP78, hemopexin, kininogen-1 and apolipoprotein E. Overall, our findings demonstrated the potential of FKC as an anti-cancer agent for the treatment of colon carcinoma.
Molecular crosstalk between the cellular epigenome and genome converge as a synergistic driver of oncogenic transformations. Besides other pathways, epigenetic regulatory circuits exert their effect towards cancer progression through the induction of DNA repair deficiencies. We explored this mechanism using a camptothecin encapsulated in β-cyclodextrin-EDTA-Fe3O4 nanoparticles (CPT-CEF)-treated HT29 cells model. We previously demonstrated that CPT-CEF treatment of HT29 cells effectively induces apoptosis and cell cycle arrest, stalling cancer progression. A comparative transcriptome analysis of CPT-CEF-treated versus untreated HT29 cells indicated that genes controlling mismatch repair, base excision repair, and homologues recombination were downregulated in these cancer cells. Our study demonstrated that treatment with CPT-CEF alleviated this repression. We observed that CPT-CEF exerts its effect by possibly affecting the DNA repair mechanism through epigenetic modulation involving genes of HMGB1, APEX1, and POLE3. Hence, we propose that CPT-CEF could be a DNA repair modulator that harnesses the cell's epigenomic plasticity to amend DNA repair deficiencies in cancer cells.
Dentatin (DEN), purified from the roots of Clausena excavata Burm f., has poor aqueous solubility that reduces its therapeutic application. The aim of this study was to assess the effects of DEN-HPβCD (hydroxypropyl-β-cyclodextrin) complex as an anticancer agent in HT29 cancer cell line and compare with a crystal DEN in dimethyl sulfoxide (DMSO). The exposure of the cancer cells to DEN or DEN-HPβCD complex leads to cell growth inhibition as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. To analyze the mechanism, in which DEN or DEN-HPβCD complex causes the death in human colon HT29 cancer cells, was evaluated by the enzyme-linked immunosorbent assay (ELIZA)-based assays for caspase-3, 8, 9, and reactive oxygen species (ROS). The findings showed that an anti-proliferative effect of DEN or DEN-HPβCD complex were via cell cycle arrest at the G2/M phase and eventually induced apoptosis through both mitochondrial and extrinsic pathways. The down-regulation of poly(ADP-ribose) polymerase (PARP) which leaded to apoptosis upon treatment, was investigated by Western-blotting. Hence, complexation between DEN and HPβCD did not diminish or eliminate the effective properties of DEN as anticancer agent. Therefore, it would be possible to resolve the conventional and current issues associated with the development and commercialization of antineoplastic agents in the future.
BACKGROUND AND PURPOSE: Andrographolide, the major phytoconstituent of Andrographis paniculata, was previously shown by us to have activity against breast cancer. This led to synthesis of new andrographolide analogues to find compounds with better activity than the parent compound. Selected benzylidene derivatives were investigated for their mechanisms of action by studying their effects on the cell cycle progression and cell death.
EXPERIMENTAL APPROACH: Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulphorhodamine B (SRB) assays were utilized in assessing the in vitro growth inhibition and cytotoxicity of compounds. Flow cytometry was used to analyse the cell cycle distribution of control and treated cells. CDK1 and CDK4 levels were determined by western blotting. Apoptotic cell death was assessed by fluorescence microscopy and flow cytometry.
KEY RESULTS: Compounds, in nanomolar to micromolar concentrations, exhibited growth inhibition and cytotoxicity in MCF-7 (breast) and HCT-116 (colon) cancer cells. In the NCI screen, 3,19-(2-bromobenzylidene) andrographolide (SRJ09) and 3,19-(3-chloro-4-fluorobenzylidene) andrographolide (SRJ23) showed greater cytotoxic potency and selectivity than andrographolide. SRJ09 and SRJ23 induced G(1) arrest and apoptosis in MCF-7 and HCT-116 cells, respectively. SRJ09 downregulated CDK4 but not CDK1 level in MCF-7 cells. Apoptosis induced by SRJ09 and SRJ23 in HCT-116 cells was confirmed by annexin V-FITC/PI flow cytometry analysis.
CONCLUSION AND IMPLICATIONS: The new benzylidene derivatives of andrographolide are potential anticancer agents. SRJ09 emerged as the lead compound in this study, exhibiting anticancer activity by downregulating CDK4 to promote a G(1) phase cell cycle arrest, coupled with induction of apoptosis.
ETHNOPHARMACOLOGICAL RELEVANCE: Annona muricata known as "the cancer killer" has been widely used in the traditional medicine for the treatment of cancer and tumors. The purpose of this study is to investigate the anticancer properties of ethyl acetate extract of Annona muricata leaves (EEAM) on HT-29 and HCT-116 colon cancer cells and the underlying mechanisms.
MATERIALS AND METHODS: The effect of EEAM on the cell proliferation of HT-29 and HCT-116 cells was analyzed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay. High content screening system (HCS) was applied to investigate the cell membrane permeability, mitochondrial membrane potential (MMP), nuclear condensation and cytochrome c translocation from mitochondria to cytosol. Reactive oxygen species (ROS) formation, lactate dehydrogenase (LDH) release and activation of caspase-3/7, -8 and -9 were measured while treatment. Flow cytometric analysis was used to determine the cell cycle distribution and phosphatidylserine externalization. The protein expression of Bax and Bcl-2 was determined using immunofluorescence analysis. In addition, the potential of EEAM to suppress the migration and invasion of colon cancer cells was also examined.
RESULTS: EEAM exerted significant cytotoxic effects on HCT-116 and HT-29 cells as determined by MTT and LDH assays. After 24 h treatment, EEAM exhibited the IC₅₀ value of 11.43 ± 1.87 µg/ml and 8.98 ± 1.24 µg/ml against HT-29 and HCT-116 cells, respectively. Flow cytometric analysis demonstrated the cell cycle arrest at G1 phase and phosphatidylserine externalization confirming the induction of apoptosis. EEAM treatment caused excessive accumulation of ROS followed by disruption of MMP, cytochrome c leakage and activation of the initiator and executioner caspases in both colon cancer cells. Immunofluorescence analysis depicted the up-regulation of Bax and down-regulation of Bcl-2 proteins while treated with EEAM. Furthermore, EEAM conspicuously blocked the migration and invasion of HT-29 and HCT-116 cells.
CONCLUSIONS: These findings provide a scientific basis for the use of A. muricata leaves in the treatment of cancer, although further in vivo studies are still required.
In situ coating of 5-fluorouracil pellets by ethylcellulose and pectin powder mixture (8:3 weight ratio) in capsule at simulated gastrointestinal media provides colon-specific drug release in vitro. This study probes into pharmacodynamic and pharmacokinetic profiles of intra-capsular pellets coated in vivo in rats with reference to their site-specific drug release outcomes. The pellets were prepared by extrusion-spheronization technique. In vitro drug content, drug release, in vivo pharmacokinetics, local colonic drug content, tumor, aberrant crypt foci, systemic hematology and clinical chemistry profiles of coated and uncoated pellets were examined against unprocessed drug. In vivo pellet coating led to reduced drug bioavailability and enhanced drug accumulation at colon (179.13 μg 5-FU/g rat colon content vs 4.66 μg/g of conventional in vitro film-coated pellets at 15 mg/kg dose). The in vivo coated pellets reduced tumor number and size, through reforming tubular epithelium with basement membrane and restricting expression of cancer from adenoma to adenocarcinoma. Unlike uncoated pellets and unprocessed drug, the coated pellets eliminated aberrant crypt foci which represented a putative preneoplastic lesion in colon cancer. They did not inflict additional systemic toxicity. In vivo pellet coating to orally target 5-fluorouracil delivery at cancerous colon is a feasible therapeutic treatment approach.
The interconnected Ras/ERK and PI3K/AKT pathways play a central role in colorectal tumorigenesis, and they are targets for elucidating mechanisms involved in attempts to induce colon cancer cell death. Both ginger (Zingiber officinale) and honey have been shown to exhibit anti-tumor and anti-inflammation properties against many types of cancer, including colorectal cancer. However, there are currently no reports showing the combined effect of these two dietary compounds in cancer growth inhibition. The aim of this study was to evaluate the synergistic effect of crude ginger extract and Gelam honey in combination as potential cancer chemopreventive agents against the colorectal cancer cell line HT29.