Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate.

Reactive oxygen species (ROS) stimulate cell proliferation and induce genetic instability, and their increase in cancer cells is often viewed as an adverse event. Here, we show that such abnormal increases in ROS can be exploited to selectively kill cancer cells using beta-phenylethyl isothiocyanate (PEITC). Oncogenic transformation of ovarian epithelial cells with H-Ras(V12) or expression of Bcr-Abl in hematopoietic cells causes elevated ROS generation and renders the malignant cells highly sensitive to PEITC, which effectively disables the glutathione antioxidant system and causes severe ROS accumulation preferentially in the transformed cells due to their active ROS output. Excessive ROS causes oxidative mitochondrial damage, inactivation of redox-sensitive molecules, and massive cell death. In vivo, PEITC exhibits therapeutic activity and prolongs animal survival.

A boronic-chalcone derivative exhibits potent anticancer activity through inhibition of the proteasome.

Chalcones and their derivatives have been shown to have potent anticancer activity. However, the exact mechanisms of cytotoxic activity remain to be established. In this study, we have evaluated a series of boronic chalcones for their anticancer activity and mechanisms of action. Among the eight chalcone derivatives tested, 3,5-bis-(4-boronic acid-benzylidene)-1-methyl-piperidin-4-one (AM114) exhibited most potent growth inhibitory activity with IC50 values of 1.5 and 0.6 microM in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay, respectively. The cytotoxic activity of AM114 was shown to be associated with the accumulation of p53 and p21 proteins and induction of apoptosis. Mechanistic studies showed that AM114 treatment inhibited the chymotrypsin-like activity of the 20S proteasome in vitro, leading to a significant accumulation of ubiquitinated p53 and other cellular proteins in whole cells. In vitro studies showed that AM114 did not significantly disrupt the interaction of p53 and murine double minute 2 protein. It is noteworthy that AM114 as a single agent was preferentially toxic to cells with wild-type p53 expression, whereas combination of this compound with ionizing radiation (IR) significantly enhanced the cell-killing activity of IR in both wild-type p53 and p53-null cells. Together, these results indicate that the boronic chalcone derivative AM114 induces significant cytotoxic effect in cancer cells through the inhibition of the cellular proteasome and provide a rationale for the further development of this class of compounds as novel cancer chemotherapeutic agents.

Anticancer activities of novel chalcone and bis-chalcone derivatives.

A series of novel chalcones and bis-chalcones containing boronic acid moieties has been synthesized and evaluated for antitumor activity against the human breast cancer MDA-MB-231 (estrogen receptor-negative) and MCF7 (estrogen receptor-positive) cell lines and against two normal breast epithelial cell lines, MCF-10A and MCF-12A. These molecules inhibited the growth of the human breast cancer cell lines at low micromolar to nanomolar concentrations, with five of them (1-4, 9) showing preferential inhibition of the human breast cancer cell lines. Furthermore, bis-chalcone 8 exhibited a more potent inhibition of colon cancer cells expressing wild-type p53 than of an isogenic cell line that was p53-null.

Novel role of p53 in maintaining mitochondrial genetic stability through interaction with DNA Pol gamma.

Mitochondrial DNA (mtDNA) mutations and deletions are frequently observed in cancer, and contribute to altered energy metabolism, increased reactive oxygen species (ROS), and attenuated apoptotic response to anticancer agents. The mechanisms by which cells maintain mitochondrial genomic integrity and the reason why cancer cells exhibit more frequent mtDNA mutations remain unclear. Here, we report that the tumor suppressor molecule p53 has a novel role in maintaining mitochondrial genetic stability through its ability to translocate to mitochondria and interact with mtDNA polymerase gamma (pol gamma) in response to mtDNA damage induced by exogenous and endogenous insults including ROS. The p53 protein physically interacts with mtDNA and pol gamma, and enhances the DNA replication function of pol gamma. Loss of p53 results in a significant increase in mtDNA vulnerability to damage, leading to increased frequency of in vivo mtDNA mutations, which are reversed by stable transfection of wild-type p53. This study provides a mechanistic explanation for the accelerating genetic instability and increased ROS stress in cancer cells associated with loss of p53.

Role of p53 in sensing oxidative DNA damage in response to reactive oxygen species-generating agents.

The tumor suppressor p53 plays an important role in the regulation of cellular response to DNA damage. Recent studies suggest that p53 is able to bind DNA with certain structural alterations in a sequence-independent manner and to interact with several molecules involved in DNA repair. This study was undertaken to test the hypothesis that p53 may participate in sensing oxidative DNA damage, the most frequently occurring spontaneous DNA lesion, and modulate its repair by the base excision repair (BER) machinery. Using synthetic DNA containing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG), we showed that p53 was pulled down together with two BER proteins, human 8-oxoguanine glycosylase (hOGG1) and AP endonuclease (APE). Functional analysis showed that p53 significantly enhanced the sequential activities of hOGG1 and APE in excising the 8-oxoG nucleotide from DNA in vitro. The ability of p53 to enhance the removal of oxidized DNA bases was further demonstrated in vivo using a pair of p53 isogenic lines. HCT116 p53+/+ cells exhibit a more rapid removal of 8-oxoG from DNA than p53-/- cells exposed to the same levels of reactive oxygen species (ROS) stress. Together, these results suggest that p53 participates in sensing oxidative DNA damage and modulates BER function in response to persistent ROS stress.

Action of (E)-2'-deoxy-2'-(fluoromethylene)cytidine on DNA metabolism: incorporation, excision, and cellular response.

(E)-2'-deoxy-2'-(fluoromethylene)cytidine (FMdC) is a new analog of deoxycytidine with promising anticancer activity. We investigated the action of FMdC on DNA metabolism by evaluating its incorporation into DNA, its excision from DNA in vitro, and the role of the incorporation of FMdC into DNA in causing cytotoxicity. In vitro DNA primer extension demonstrated that FMdC nucleotides were incorporated with relatively high substrate efficiency into the C sites of the elongating DNA strand. Once incorporated, FMdC became a poor substrate for further chain elongation by DNA polymerases, resulting in a termination of DNA synthesis at the sites of incorporation. Furthermore, the 3' --> 5' exonuclease activity of DNA polymerase epsilon or wild-type p53 protein was ineffective in removing the incorporated FMdC from DNA in vitro. FMdC also showed potent cytotoxic activity against human leukemia and solid tumor cells. Incubation with a low concentration of FMdC (10 nM) induced cell cycle arrest at S or G1 phases, but the cells eventually died as the time of incubation increased. Compared with HL-60 cells, human myeloid ML-1 cells with wild-type p53 were more sensitive to FMdC, but the S or G1 phase arrest did not seem to depend on the presence or absence of p53. Inhibiting the incorporation of FMdC into cellular DNA by aphidicolin suppressed the cytotoxic effect of the compound. We conclude that the incorporated FMdC nucleotide profoundly disrupts DNA synthesis and resists excision by exonucleases, and that incorporation of this analog into DNA is a key molecular event responsible for the drug's cytotoxicity.

Superoxide dismutase: an emerging target for cancer therapeutics.

Superoxide dismutase (SOD) is a critical enzyme responsible for the elimination of superoxide radicals and is considered to be a key anti-oxidant in aerobic cells. Cellular consumption of oxygen is essential for oxidative phosphorylation during ATP generation in the mitochondria, yet this cellular metabolism also leads to the production of reactive oxygen species (ROS), including the superoxide radical (O(2)(*)(-)) and hydrogen peroxide (H(2)O(2)). Accumulation of ROS results in cellular oxidative stress and, if not corrected, can lead to the damage of important biomolecules such as membrane lipids, proteins and DNA. Prolonged accumulation of high levels of free radicals in cells may cause irreversible cellular injury and ultimately result in cell death. Since SOD is the key enzyme in the first metabolic step of superoxide elimination, deficiency in SOD or inhibition of the enzyme activity may cause severe accumulation of O(2)(*)(-) in cells and lead to cell death. Thus, inhibition of SOD may provide a novel way to kill cancer cells. Due to dysfunction in the regulation of cell growth, cancer cells are active in energy metabolism, and thus produce high levels of O(2)(*)(-) and other ROS and are under constant oxidative stress. This may render the malignant cells more dependent on SOD to eliminate the toxic superoxide radicals and thus potentially more sensitive to SOD inhibitors. It is a plausible hypothesis that inhibition of SOD may preferentially kill malignant cells through a free radical-mediated mechanism. This article will review evidence that suggests SOD as an emerging therapeutic target for cancer treatment. The relevant clinical implications and potential risk will also be discussed.