Apoptosis In COLO Research Articles

Molecular mechanisms of denbinobin-induced anti-tumorigenesis effect in colon cancer cells

To explore both the in vitro and in vivo effects of denbinobin against colon cancer cells and clarify its underlying signal pathways. We used COLO 205 cancer cell lines and nude mice xenograft model to study the in vitro and in vivo anti-cancer effects of denbinobin. Denbinobin at concentration of 10-20 micromol/L dose-dependently suppressed COLO 205 cell proliferation by MTT test. Flow cytometry analysis and DNA fragmentation assay revealed that 10-20 micromol/L denbinobin treatment induced COLO 205 cells apoptosis. Western blot analysis showed that caspases 3, 8, 9 and Bid protein were activated by denbinobin treatment to COLO 205 cells accompanied with cytochrome c and apoptosis-inducing factor (AIF) translocation. Pretreatment of MEK 1 inhibitor (U10126), but not p38 inhibitor (SB203580) and JNK inhibitor (SP600125), reversed denbinobin-induced caspase 8, 9 and Bid activation in COLO 205 cells suggesting that extracellular signal-regulated kinase were involved in the denbinobin-induced apoptosis in COLO 205 cells. Significant regression of tumor up to 68% was further demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with denbinobin 50 mg/kg intraperitoneally. Our findings suggest that denbinobin could inhibit colon cancer growth both in vitro and in vivo. Activation of extrinsic and intrinsic apoptotic pathways and AIF were involved in the denbinobin-induced COLO 205 cell apoptosis.

Evidence Supporting a Role for Calcium in Apoptosis Induction by the Synthetic Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic Acid (CDDO)

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a novel anticancer agent that induces apoptosis in tumor cells. The cytotoxic stress underpinning CDDO-induced apoptosis has not been established. This study compared and contrasted the effects of CDDO on COLO 16 human skin cancer cells and their respiration-deficient (rho(0)) clones to elucidate the stress signal responsible for initiating apoptosis. CDDO promoted apoptosis in COLO 16 cells in a dose- and time-dependent manner. The rho(0) clones appeared to be more sensitive to CDDO-induced apoptosis implying that the disruption of mitochondrial respiration was not directly associated with triggering cell death. After a 4-h exposure to CDDO, mitochondrial inner transmembrane potential-sensitive dyes revealed mitochondrial hyperpolarization in the COLO 16 cells and mitochondrial depolarization in the rho(0) clones. Electron microscopy illustrated that this exposure also promoted mitochondrial condensation, endoplasmic reticulum dilation, and chromatin condensation in the COLO 16 cells. Endoplasmic reticulum dilation and chromatin condensation were also observed in the rho(0) clones, but the mitochondria in these cells were markedly swollen implying that the disruption of intracellular Ca(2+) homeostasis was associated with cell death. A Ca(2+)-sensitive dye confirmed that CDDO increased cytoplasmic free Ca(2+) in the COLO 16 cells, their rho(0) clones, as well as in malignant breast and lung epithelial cells. A cell-permeant Ca(2+) chelator reduced the CDDO-induced increase in cytoplasmic free Ca(2+), and inhibited caspase activation, the development of apoptotic morphology, and DNA fragmentation in the COLO 16 cells, implying that Ca(2+) played a pivotal role in signaling the initiation of apoptosis.

Protein kinase C inhibition induces DNA fragmentation in COLO 205 cells which is blocked by cysteine protease inhibition but not mediated through caspase-3

Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([ 3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway.

Elimination of extrachromosomal c-myc genes by hydroxyurea induces apoptosis.

Hydroxyurea (HU) increases extrachromosomal DNA elimination in tumor cell lines. The c-myc oncogene is one of the many relevant amplified genes contained within the extrachromosomal DNA compartment. Spontaneous loss of amplified copies of c-myc induces terminal differentiation and apoptosis in the human HL-60 leukemia cell lines. In the present study, we evaluate HU's ability to induce apoptosis by eliminating extrachromosomally located c-myc oncogene in human tumor cell lines. The consequences of eliminating extrachromosomal DNA by HU were explored in two different cell lines using the TdT assay and acridine orange/ethidium bromide labeling. COLO 320 clone 3 and COLO 320 clone 21 cell lines contain the same number of amplified copies of c-myc oncogene, but located respectively on extrachromosomal DNA, and intrachromosomally in homogeneously staining regions. HU induced apoptosis in the COLO 320 clone 3 cell line by a time and concentration dependent mechanism but could not induce apoptosis in the COLO 320 clone 21 cell line. These results suggested that HU-induced apoptosis in COLO 320 cell lines depends on elimination of extrachromosomal amplified copies of the c-myc oncogene. The ability of HU to eliminate extrachromosomally amplified copies of the c-myc oncogene and to induce apoptosis should be considered when targeting malignancies with amplification of the c-myc oncogene in an extrachromosomal site.

Mechanisms Underlying Apoptosis Induced by Combination of 5-Fluorouracil and Interferon-γ

Several investigators have reported on the clinical effects of 5-fluorouracil (5-FU) or the combination of 5-FU plus interferon-γ (IFN-γ) on patients with advanced colorectal carcinoma. It has also been reported that apoptosis induced by 5-FU is due to the effects on DNA synthesis and functional RNA synthesis. In the present study, we examine the biological mechanisms underlying 5-FU or the combination of 5-FU plus IFN-γ, using the colorectal carcinoma cell line, COLO 201. 5-FU and IFN-γ independently or additively induced apoptosis in COLO 201 in a dose- and time-dependent manner, which was correlated with the down-regulation of Bcl-2 and the up-regulation of Bax. An interleukin-1β-converting enzyme (ICE)-like protease inhibitor (but not an ICE-inhibitor) blocked apoptosis induced by only 5-FU. These results suggest that 5-FU has the capacity to induce apoptosis in COLO 201, resulting from the up-regulation of Bax; the apoptosis-inducing signal of 5-FU seems to be different from that of IFN-γ. Thereby, 5-FU and IFN-γ have additional effects on the induction of apoptosis. This finding provides an experimental basis for clinical therapy using 5-FU and/or IFN-γ for colorectal cancer.

Suppression of nitric oxide–induced apoptosis by N-acetyl-l-cysteine through modulation of glutathione, bcl-2, and bax protein levels

It has been demonstrated that nitric oxide (NO) can promote apoptosis in human cancer cells. To test the protective effects of antioxidants (N-acetyl-L-cysteine (LNAC) and free-radical spin traps (5,5-dimethyl-1-pyrroline N-oxide and 2,2,6,6,-tetramethyl-1-piperidinyloxy) against NO-induced apoptosis, a human colon cancer cell line (COLO 205) was treated with NO, and its survival rate was evaluated both with and without antioxidant therapy. LNAC arrested the development of progression of apoptosis in COLO 205 cells in a dose-dependent manner, promoted long-term survival, and prevented the internucleosomal DNA cleavage induced by NO. The intracellular level of glutathione (GSH) was found to be elevated in cells after exposure to LNAC. The bax protein levels were elevated by NO treatment, and this effect was blocked by LNAC. On the other hand, the bcl-2 oncoprotein level in the LNAC-pretreated cells was significantly elevated in a time-dependent manner compared to cells that received NO pretreatment. In summary, our results suggest that the protective effect of LNAC may be linked to its inducement of increases in cellular GSH and bcl-2 protein levels and to its suppression of cellular bax protein in treated cells.

Suppression of apoptosis in COLO 205 cells by the phorbol ester TPA may be mediated by the PKC isoenzyme alpha

Apoptosis induced by an antibody to CD95/APO-1/FAS in the colon carcinoma cells COLO 205 and HT-29 is suppressed by the phorbol ester TPA. Inhibition is much more effective in COLO 205 than in HT-29 cells. The TPA effect is abrogated by the protein kinase C (PKC)-specific inhibitor Go6983 indicating a role of PKC in this process. Bryostatin 1, unlike TPA, is unable to suppress apoptosis, but inhibits the TPA-induced suppression of apoptosis. TPA also inhibits indomethacin-induced apoptosis in COLO 205 cells. COLO 205 and HT-29 cells contain the PKC isoenzymes alpha, beta(II) delta, epsilon, eta, mu and zeta. Expression and activity of PKC alpha are at least 5 times higher in COLO 205 than in HT-29 cells. This correlates with the fact that inhibition of CD95-mediated apoptosis by TPA is more prominent in COLO 205 than in HT-29 cells. Thus, these findings suggest that PKC alpha has an important role in the TPA-induced inhibition of apoptosis.