mtt-formazan and Colorectal-Neoplasms

Cancer is the second leading cause of death worldwide, the deaths are projected to continue rising, with an estimated 12 million deaths in 2030. The aim of the present investigation is to prepare and compare the uncoated (U-CH NP) and eudragit S 100-coated (E-U-CH NP) chitosan nanoparticles encapsulating a caspase 3 activator (UCN 01), by ionic gelation method. The prepared formulations were studied for various parameters like particles size, zeta potential, transmission electron microscopy, atomic force microscopy, in vitro release study, ex vivo study using Caco 2 colon cancer cell line, and in vivo studies. The particle size and zeta potential of developed formulation was found to be particle size of 168 ± 3.7 nm and +35.8 ± 3.7 for U-CH NP and 265 ± 4.1 nm and +22.3 ± 1.1 for E-U-CH NP. TEM and AFM images revealed that U-CH NPs were round in shape and smoother at surface as compared to E-U-CH NP which have irregular surface due to coating. The E-U-CH NP showed better in vitro release than uncoated formulation in SCF (pH 6.8) than in SGF (pH 1.2). The cytotoxicity was performed by MTT assay. U-CH NP showed enhanced cytotoxicity as compared to blank (without drug) formulation. There was an increase in caspase 3 activity of U-CH NP as compared to UCN 01 alone. E-U-CH NP showed better tumor regression ability than U-CH NP. The results of plasma profile and tumor regression study demonstrated that E-U-CH NP has continuous release profile of UCN 01 and comprehensive residence time. Thus, it is better acceptable than free UCN 01 and may be a potential delivery system for the targeting and treatment of colon cancer.

Topics: Animals; Antineoplastic Agents; Caco-2 Cells; Caspase 3; Chitosan; Colorectal Neoplasms; Drug Carriers; Drug Delivery Systems; Enzyme Activation; Formazans; Humans; Nanoparticles; Particle Size; Polymethacrylic Acids; Rabbits; Staurosporine; Tetrazolium Salts

Arsenic trioxide (As2O3) shows a significant therapeutic effect upon acute promyelocytic leukemia (APL) and can induce the apoptosis of NB4 cells, which attracts scholars' great attention. Especially, the therapeutic effect on solid carcinoma has been paid more close attention to. The present study is to evaluate the effect of As2O3 on human colorectal carcinoma cells (LS-174T cell) and the activity of telomerase in vitro and in vivo. This research made use of the electron microscope, polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), fluorescence-activated cell sorter (FACS), MTT in vitro and in vivo (LS-174T xenograft model of nude mice). With the increasing concentration of As2O3, the ratio of living cells to dead cells decreased significantly, and the IC50 value was 5.23micromol/L; cells of the experimental groups endured a series of morphological changes similar to the features of apoptosis. Apoptosis curve of FACS pictures appeared after 24h, and the cells showed apoptosis in a time-dependent manner; As2O3 can inhibit the activity of telomerase of the cell extraction, obviously, in a concentration-dependent and time-dependent manner after 24h. As to the inhibition impact of As2O3 on the xenograft model of nude mice in the two indexes, tumor volume and weight, there was a significant difference between As2O3 and the control group; there was no difference between As2O3 and the fluorouracil (5-FU) group; in the group of peritoneal injections of As2O3, the cancer cells connected loosely with each other, nucleus changed markedly, and heterochromatin concentrated under the nucleus membrane. From the in vitro and in vivo experiment, we can see that As2O3 inhibited LS-174T cell growth mainly by inducing cell apoptosis, partly by the inhibition of telomerase activity.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Line, Tumor; Cell Nucleus; Cell Survival; Colorectal Neoplasms; Cytoplasmic Structures; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Formazans; Humans; Mice; Mice, Nude; Oxides; Polymerase Chain Reaction; Telomerase; Tetrazolium Salts; Xenograft Model Antitumor Assays

Polyamines have been shown to play a role in the growth and survival of several solid tumors, including colorectal cancer. We identified the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as being one of the most highly inducible genes in two DNA microarray screens to identify novel determinants of response to chemotherapeutic agents in colorectal cancer. SSAT was shown to be inducible in response to 5-fluorouracil (5-FU) or oxaliplatin in parental and drug-resistant HCT116 cell lines. It was also shown that SSAT mRNA was up-regulated in response to 5-FU or oxaliplatin in a panel of six colorectal cancer cell lines. The polyamine analogue N(1),N(11)-diethylnorspermine (DENSpm) depletes polyamine pools and potently induces SSAT. We evaluated the effect of combining DENSpm with chemotherapeutic agents in HCT116 p53(+/+) cells and in HCT116 drug-resistant daughter cell lines. Western blot analyses showed that SSAT protein expression was dramatically enhanced when DENSpm was combined with oxaliplatin or 5-FU in HCT116 p53(+/+) cells. Using cell viability assays and flow cytometry, synergistic induction of cell death was observed following cotreatment of HCT116 p53(+/+) cells with DENSpm and each chemotherapeutic agent. Of note, this combined therapy increased the chemosensitivity of cells rendered resistant to each of these chemotherapeutic agents. Small interfering RNA-mediated down-regulation of SSAT resulted in loss of synergy between DENSpm and these agents. These results show that SSAT plays an important role in regulating cell death following combined cytotoxic drug and DENSpm treatment. Furthermore, DENSpm sensitizes both sensitive and resistant cells to chemotherapeutic agents. Taken together, these results suggest that SSAT may be an important target for therapeutic intervention in colorectal cancer.

Topics: Acetyltransferases; Antineoplastic Agents; Cell Death; Cell Survival; Colorectal Neoplasms; Drug Resistance, Neoplasm; Drug Synergism; Formazans; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; RNA, Messenger; RNA, Small Interfering; Spermine; Tetrazolium Salts; Tumor Suppressor Protein p53