Barbara A. Woynarowska

DNA strand breaks and apoptosis induced by oxaliplatin in cancer cells.

Platinum anticancer drugs, such as cisplatin, are thought to exert their activity by DNA damage. Oxaliplatin, a clinically active diaminocyclohexane platinum compound, however, requires fewer DNA-Pt adducts than cisplatin to achieve cell growth inhibition. Here we investigated whether secondary DNA damage and apoptotic responses to oxaliplatin compensate for the reduced formation of DNA adducts. Oxaliplatin treatment of leukemic CEM and ovarian A2780 cancer cells resulted in early (4 hr) induction of DNA single-strand breaks measured by nucleoid sedimentation. These infrequent early lesions progress with time into massive double-stranded DNA fragmentation (fragments >50k bp) paralleled by characteristic apoptotic changes revealed by cell morphology and multivariate flow cytometry. Profound oxaliplatin-induced apoptotic DNA fragmentation was detectable following a 24 hr treatment of A2780 and CEM cells with 2 and 10 microM oxaliplatin, respectively. This DNA fragmentation was inhibited completely by the broad-spectrum caspase inhibitor Z-VAD-fmk. Cisplatin, which forms markedly more DNA-Pt adducts in CEM and A2780 cells than equimolar oxaliplatin, was similarly potent as oxaliplatin in terms of early strand breaks and later apoptotic responses. Oxaliplatin was also profoundly apoptotic in several other tumor cell lines of prostate origin but had only a marginal effect in normal prostate PrEC cells. Collectively, the results demonstrate that, relative to the magnitude of the primary DNA-Pt lesions, oxaliplatin is disproportionately more potent than cisplatin in the induction of apoptosis. Apoptosis induction, possibly enhanced by a contribution of targets other than DNA, seems to be an important factor in the mechanism of action of oxaliplatin.

Differential cytotoxicity and induction of apoptosis in tumor and normal cells by hydroxymethylacylfulvene (HMAF)

This investigation compared the effects of hydroxymethylacylfulvene (HMAF), a novel antitumor drug with alkylating properties, in eight human tumor (prostate, colon, and leukemia) cell lines, and five human normal (prostate and renal proximal tubule epithelial, colon mucosa, fibroblasts, and endothelial) cell lines. Drug-induced growth inhibition paralleled the uptake of HMAF into both tumor and normal cells, although normal cells were 3- to 4-fold more tolerant to the accumulated drug. In both tumor and normal cells, approximately two-thirds of internalized [(14)C]HMAF-derived radioactivity was bound covalently to macromolecules. Trypan blue exclusion and cell counts indicated that HMAF was cytotoxic in tumor but cytostatic in normal cells. Correspondingly, profound apoptosis was detected in all tumor cell lines examined. A 4-hr treatment with HMAF followed by 20-hr post-incubation induced a potent DNA fragmentation in nearly all tumor lines. Apoptosis-resistant PC-3 and HT-29 cells underwent significant DNA fragmentation after 24 hr of continuous treatment with HMAF. In contrast to tumor cell lines, marginal or very low levels of apoptosis were detected in the normal cells even after prolonged treatments with HMAF at concentrations that exceeded 15- to 800-fold the GI(50) values in tumor cells. This resistance of normal cells to apoptosis could not be accounted for by differences in drug accumulation or drug covalent binding to macromolecules. The qualitatively different responses of the tumor and normal cells studied suggest a greater tolerance of normal cells to HMAF-macromolecular adducts. The demonstrated differential cytotoxic/cytostatic and apoptotic effects of HMAF can be of significance for the clinical use of this promising new agent.

Targeting Apoptosis by Hydroxymethylacylfulvene in Combination with Gamma Radiation in Prostate Tumor Cells

Abstract Woynarowska, B. A., Roberts, K., Woynarowski, J. M., MacDonald, J. R. and Herman, T. S. Targeting Apoptosis by Hydroxymethylacylfulvene in Combination with Gamma Radiation in Prostate Tumor Cells. Hydroxymethylacylfulvene (HMAF) is a novel agent with alkylating activity and is a potent inducer of apoptosis that is currently undergoing Phase II clinical trials for prostate cancer. This study explored the pro-apoptosis and anti-proliferative potential of HMAF in combination with γ radiation in human prostate tumor cell lines. Apoptosis was assessed based on the generation of fragmented DNA, a terminal transferase flow cytometry assay, and cell morphology. In each of the tumor cell lines examined, radiation alone induced a marginal level of apoptosis, even after a prolonged 48-h incubation after exposure. In contrast, HMAF alone was a potent inducer of apoptosis in prostate tumor cells but not in normal cells. Marked levels of apoptosis in tumor cells were also observed for the combination of HMAF with γ radiation. When drug treatment preceded irradiation, at least additive levels of apoptosis were observed in both androgen-responsive and androgen-independent cells. The combined treatment with ionizing radiation and HMAF reduced the radiation dose needed for the same level of clonogenic survival up to 2.5-fold. The potentiation of apoptosis and reduction in the clonogenic survival of tumor cells occurred at HMAF concentrations lower than that which reduced survival to 10% and at doses up to 6 Gy. No potentiation of apoptosis or clonogenic inhibition was noted in normal cells. These results suggest that the combination of HMAF with γ radiation may have clinical utility for treatments of prostate cancer.

Apoptosis induction by the dual-action DNA- and protein-reactive antitumor drug irofulven is largely Bcl-2-independent

The overexpression of Bcl-2 is implicated in the resistance of cancer cells to apoptosis. This study explored the potential of irofulven (hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863), a novel DNA- and protein-reactive anticancer drug, to overcome the anti-apoptotic properties of Bcl-2 in HeLa cells with controlled Bcl-2 overexpression. Irofulven treatment resulted in rapid (12hr) dissipation of the mitochondrial membrane potential, phosphatidylserine externalization, and apoptotic DNA fragmentation, with progressive changes after 24hr. Bcl-2 overexpression caused marginal or partial inhibition of these effects after treatment times ranging from 12 to 48hr. Both Bcl-2-dependent and -independent responses to irofulven were abrogated by a broad-spectrum caspase inhibitor. Despite the somewhat decreased apoptotic indices, cell growth inhibition by irofulven was unaffected by Bcl-2 status. In comparison, Bcl-2 overexpression drastically reduced apoptotic DNA fragmentation by etoposide, acting via topoisomerase II-mediated DNA damage, but had no effect on apoptotic DNA fragmentation by helenalin A, which reacts with proteins but not DNA. Irofulven retains its pro-apoptotic and growth inhibitory potential in cell lines that have naturally high Bcl-2 expression. Collectively, the results implicate multiple mechanisms of apoptosis induction by irofulven, which may differ in time course and Bcl-2 dependence. It is possible that the sustained ability of irofulven to induce profound apoptosis and to block cell growth despite Bcl-2 overexpression may be related to its dual reactivity with both DNA and proteins.

Irofulven Induces Apoptosis in Breast Cancer Cells Regardless of Caspase-3 Status*

Caspase-3 deficiency can limit the efficiency of pro-apoptotic anticancer treatments. Irofulven (hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863) is an antitumor drug, currently in a Phase III and multiple Phase II trials, which can differentiate between tumor and normal cells in apoptosis induction. This study investigated whether apoptosis induced by irofulven requires caspase-3. Irofulven action was compared in breast cancer cells differing in caspase-3 status: deficient MCF-7 cells and proficient MDA-MB-231 cells and in normal human mammary epithelial cells, HMEC. Irofulven induces significant, concentration and time-dependent apoptotic DNA fragmentation in breast cancer cell lines, regardless of caspase-3 status. After 12, 24 and 48 h incubation at 1 μM irofulven (∼ 3 × GI50), fragmented DNA comprised 3.7, 14.1 and 34.6% and 8.4, 12.6 and 20.3% of total DNA in MCF-7 and MDA-MB-231 cells, respectively. Cell viability (trypan blue exclusion) remained largely unaffected during the first 24 h but decreased markedly after 48 h, indicating secondary necrosis. Net losses in cell numbers were apparent at 48 h. Normal HMEC cells were refractory to 1 μM drug with only ∼3–9% fragmented DNA after 12–48 h, although apoptosis was observed at drug levels >3 μM. The broad-spectrum caspase inhibitor Z-VAD-fmk inhibited irofulven-induced apoptosis of all cell lines at 20 μM with nearly complete abrogation of apoptosis at 100 μM. Irofulven treatment resulted in marginal caspase-3 processing in MDA-MB-231 and HMEC cells. These results indicate that whereas the caspase cascade mediates irofulven- induced apoptosis, caspase-3 is dispensable (supported by NIH CA70091 and CA78706).

WMC-79, a potent agent against colon cancers, induces apoptosis through a p53-dependent pathway

WMC-79 is a synthetic agent with potent activity against colon and hematopoietic tumors. In vitro, the agent is most potent against colon cancer cells that carry the wild-type p53 tumor suppressor gene (HCT-116 and RKO cells: GI50 <1 nmol/L, LC50 ∼40 nmol/L). Growth arrest of HCT-116 and RKO cells occurs at the G1 and G2-M check points at sublethal concentrations (10 nmol/L) but the entire cell population was killed at 100 nmol/L. WMC-79 is localized to the nucleus where it binds to DNA. We hypothesized that WMC-79 binding to DNA is recognized as an unrepairable damage in the tumor cells, which results in p53 activation. This triggers transcriptional up-regulation of p53-dependent genes involved in replication, cell cycle progression, growth arrest, and apoptosis as evidenced by DNA microarrays. The change in the transcriptional profile of HCT-116 cells is followed by a change in the levels of cell cycle regulatory proteins and apoptosis. The recruitment of the p53-dependent apoptosis pathway was suggested by the up-regulation of p53, p21, Bax, DR-4, DR-5, and p53 phosphorylated on Ser15; down-regulation of Bcl-2; and activation of caspase-8, -9, -7, and -3 in cells treated with 100 nmol/L WMC-79. Apoptosis was also evident from the flow cytometric studies of drug-treated HCT-116 cells as well as from the appearance of nuclear fragmentation. However, whereas this pathway is important in wild-type p53 colon tumors, other pathways are also in operation because colon cancer cell lines in which the p53 gene is mutated are also affected by higher concentrations of WMC-79.

Pilocarpine, a salivary gland radioprotectant, does not inhibit cytotoxic effect of γ-radiation on squamous cell carcinoma in vitro

PURPOSE Pilocarpine, a salivary stimulant, has been shown to protect salivary glands from gamma-radiation-induced damage during the radiotherapy of head and neck tumors. This study was performed to determine whether pilocarpine affects the survival of squamous carcinoma cells, line SCC-25, following gamma-radiation treatment. METHODS AND MATERIALS The survival of squamous carcinoma tumor cells, line SCC-25, following the exposure of cells to pilocarpine at concentration of 0-100 ng/ml given for 0-1 h prior to radiation at dose of 0-20 Gy was determined by an in vitro colony-formation assay. RESULTS The survival fractions of SCC-25 cells were identical for the control and pilocarpine-treated samples at all tested conditions. Calculated Do and Dq values did not depend on the presence of pilocarpine and were not affected by the time of incubation prior to irradiation. CONCLUSION Pilocarpine, at clinically relevant concentrations, given to the SCC-25 cells 1 h prior to or at the time of irradiation did not affect survival of SCC-25 cells in vitro. Pilocarpine does not sensitize or protect these tumor cells from the effects of y-radiation, suggesting that this agent should not compromise the tumoricidal effects of radiotherapy.

Changes in Prostate-Specific Antigen (PSA) Level Correlate with Growth Inhibition of Prostate Cancer Cells Treated in Vitro with a Novel Anticancer Drug, Irofulven

Irofulven (hydroxymethylacylfulvene,HMAF, MGI 114) is a novel agent withalkylating activity and a potent inducer ofapoptosis. It is currently undergoing PhaseII clinical trials for several tumor types,including hormone-refractory prostatecancer. Reduction of serumprostate-specific antigen (PSA) levels hasbeen proposed as a generally usefulendpoint for evaluating the antitumorefficacy of treatments for prostate cancer.However, the utility of PSA as a marker oftumor cell burden could be compromised, ifdrugs directly affected PSA secretionand/or expression. In these studies, weevaluated the effects of irofulven on PSAprotein and mRNA levels during the courseof treatment of prostate tumor cells in vitro. Therate of PSA secretion(normalized per equal cell number) bycontrol and drug treated cells was similar,as determined by a solid phase, two-siteimmunoradiometric assay. Consistent withthe lack of effect of irofulven on PSAprotein level, the drug does not appear toaffect the expression of PSA mRNA (on a percell basis) as assessed by RT-PCR. Thus,changes in PSA secretion and expressionappear to reflect irofulven-induced cellgrowth inhibition rather than reflecting adirect effect of the drug on PSA. Theseresults suggest that PSA should be areasonable marker of tumor burden inirofulven-treated prostate cancerpatients.

The Significance of Poly-Targeting in Apoptosis Induction by Alkylating Agents and Platinum Drugs

Apoptosis is believed to be an important aspect of the anticancer potency of alkylating agents (AAs) and platinum (Pt) complexes. Despite the high clinical utility of these classes of drugs, the nature and determinants of the apoptotic sensitivity/resistance of cancer cells to these agents are not completely understood. One underappreciated aspect is the wide and variable spectrum of cellular targets of AAs and Pt drugs and the complexity of the responses to poly-targeted insults. This chapter discusses the heterogeneity of targeting profiles for diverse drug types and the interdependence of apoptotic routes elicited by the damage to various cellular targets. Although many of these agents target DNA, DNA damage is not the only cause of their apoptotic effects. Drugs that alkylate proteins are strongly apoptotic, even if they do not react with DNA. The ability of alkylating and Pt drugs to damage and inactivate specific proteins and to globally distort the state of the proteome needs to be considered as a self-standing apoptotic stimulus and a factor that enhances lethal responses to DNA damage. Particular emphasis is placed on the significance of drug effects on redox-regulating proteins of the thioredoxin family. Disruption of protein redox homeostasis is likely to be critical for death/survival in response to poly-targeted alkylating and Pt drugs. Differential distortion of redox regulation is suggested as a molecular basis underlying the demonstrated potential of specific drugs such as irofulven and oxaliplatin to promote apoptosis in cancer cells while sparing normal cells.