Sulforaphane increases the efficacy of doxorubicin in mouse fibroblasts characterized by p53 mutations.

One novel strategy for increasing cancer chemotherapy efficacy and reversing chemoresistance involves co-administration of natural chemopreventive compounds alongside standard chemotherapeutic protocols. Sulforaphane is a particularly promising chemopreventive agent, which has been shown to exert proapoptotic effects on tumor cells containing p53 mutations. The p53(Ser220) mutation has been implicated in reduced efficacy and drug resistance in the context of osteosarcomas and breast tumors treated with doxorubicin-based protocols. We investigated the effects of a combination of doxorubicin and sulforaphane on cell viability and apoptosis induction in fibroblasts characterized by different p53 status (p53 wild-type, p53 knock-out, and p53(Ser220) mutation), and identified some of the molecular pathways triggered by the drug combination. Very high concentrations of doxorubicin were necessary to decrease the viability of p53(Ser220) and p53 knock-out (but not wild-type) cells. Treatment of p53(Ser220) and p53 knock-out cells with doxorubicin did not induce apoptosis, also at very high concentrations (10muM). Sulforaphane restored chemosensitivity and induced apoptosis in doxorubicin-resistant p53(Ser220) and p53 knock-out cells, irrespective of p53 status. The induction of apoptosis was caspase-3 dependent and caspase-8 independent. Bongkrekic acid, a mitochondrial membrane stabilizer, partially prevented the effects of doxorubicin plus sulforaphane on mitochondrial permeability but was unable to prevent the induction of apoptosis. N-acetyl-cysteine, a glutathione precursor, blocked the induction of apoptosis by doxorubicin plus sulforaphane. Considering the negligible safety profile of sulforaphane, our findings could prompt innovative clinical studies designed to investigate whether its coadministration can enhance the efficacy of doxorubicin-based regimens.

In vitro antitumor activity of cyanidin-3-O-beta-glucopyranoside.

BACKGROUND: Little is currently known regarding the cancer-preventive potential of cyanidin-3-O-beta-glucopyranoside (Cy-g) apart from its antioxidant activity. METHODS: We tested Cy-g on Jurkat and HL-60 leukemia cell lines and, to help elucidate whether the effects of Cy-g are specific for cancer cells, also on normal T lymphocytes. RESULTS: Cy-g induced apoptosis in all three cell systems, and this indicated that Cy-g was not selective towards leukemia cells. Moreover, Cy-g caused HL-60 differentiation. The induction of apoptosis and cytodifferentiation involved different proteins, thus suggesting that Cy-g-induced apoptosis and cytodifferentiation are two distinct events. CONCLUSIONS: Although obtained in vitro, our findings indicate that Cy-g possesses some interesting biological properties that should encourage further investigation as regards its chemotherapeutic potential.

In vitro anticancer activity of cyanidin-3-O-beta-glucopyranoside: effects on transformed and non-transformed T lymphocytes.

BACKGROUND: Little is known regarding the potentially chemopreventive activity of cyanidin-3-O-beta-glucopyranoside (Cy-g), the main anthocyanin present in the juice of pigmented oranges, apart from its antioxidant activity. After excluding a potential genotoxicity of Cy-g, its ability to induce apoptosis on transformed and normal T cells was analysed. In order to delineate the events leading to apoptosis, the expression of different proteins, known to be involved in apoptosis, was also measured. MATERIALS AND METHODS: The evaluation of genotoxicity was performed by the micronucleus test. Flow cytometry was used for the analysis of apoptotic cells and proteins involved in the modulation of apoptosis. RESULTS: Cy-g was nongenotoxic. Moreover, it induced apoptosis in both cell systems, modulated by an increase of p53 and bax proteins. CONCLUSION: These interesting biological properties should encourage further studies into the chemopreventive potential of Cy-g. Nevertheless, its activity in normal T cells underlines the need for extensive toxicological investigation.

A mutated p53 status did not prevent the induction of apoptosis by sulforaphane, a promising anti-cancer drug.

We investigated apoptosis induction by sulforaphane on three cell lines characterized by a different p53 status. In particular, we used p53-knock-out fibroblasts from newborn mice transfected with the p53-Ser220 mutation, observed in Li-Fraumeni Syndrome patients, as a model of mutated p53 status. Moreover, immortalized fibroblasts from newborn mice expressing or lacking p53 (p53 +/+ and p53-/-, respectively) have been used to verify whether mutated p53 status could prevent sulforaphane-induced apoptotic events. Sulforaphane was able to induce apoptosis on all three cell lines. Indeed, the caspase-3 assays and poly(ADP-ribose)polymerase (PARP) cleavage data indicated that sulforaphane stimulated caspase-3-like activity and degradation of PARP. However, cells with a wild-type or mutated p53 appeared to be more sensitive to the effects of sulforaphane than cells lacking p53. Taken together, our results suggest that sulforaphane could act by a p53-independent pathway. For this reason, sulforaphane can be viewed as a novel agent useful not only in the treatment of Li-Fraumeni-associated tumors but also drug resistant tumors where p53 dysregulation is a feature.

Isothiocyanates as novel cytotoxic and cytostatic agents: molecular pathway on human transformed and non-transformed cells.

Cancer chemoprevention is a new approach in the management of cancer. Traditional cytotoxic chemotherapeutic approaches cannot cure most advanced solid malignancies. Chemoprevention can be defined as the use of non-cytotoxic drugs and natural agents to block the progression to invasive cancer. Recently, isothiocyanates, natural products found in the diet of humans, has been shown to function as cancer chemopreventive agents. They are strong inhibitors of phase I enzymes and inducers of phase II enzymes. They can also induce apoptosis and modulate cell-cycle progression of highly proliferating cancer cells. This commentary will review the mechanism of apoptosis and growth inhibition mediated by different isothiocyanates. Particular attention will be given to the effects of the new isothiocyanate 4-(methylthio)butylisothiocyanate (MTBITC). Since selective targeting and low toxicity for normal host tissues are fundamental requisites for proposed chemopreventive agents, we will also review the effects of different isothiocyanates on non-transformed human cells.

Induction of apoptosis in two human leukemia cell lines as well as differentiation in human promyelocytic cells by cyanidin-3-O-beta-glucopyranoside.

Little is known about the potentially chemopreventive mechanisms of anthocyanins apart from their antioxidant activity. We investigated the in vitro capacity of the anthocyanin cyanidin-3-O-beta-glucopyranoside (Cy-g) to induce apoptosis in T-lymphoblastoid, as well as apoptosis and differentiation in HL-60 promyelocytic cells. Although Cy-g-induced apoptosis (as well as necrosis) in the two systems, HL-60 cells were much less sensitive than T-lymphoblastoid cells. Moreover, treatment of HL-60 cells with Cy-g caused differentiation into macrophage-like cells and granulocytes. Concerning the mechanism of action, the induction of apoptosis in Jurkat T cells can be explained by a modulation of p53 and bax protein expression. At the molecular level, the induction of apoptosis and cytodifferentiation in HL-60 cells involved different proteins, thus suggesting that the effects of Cy-g on apoptosis and cytodifferentiation induction are two distinct events. These interesting biological properties should encourage further investigation into the chemopreventive and/or chemotherapeutic potential of Cy-g.

The new isothiocyanate 4-(methylthio)butylisothiocyanate selectively affects cell-cycle progression and apoptosis induction of human leukemia cells.

We investigated proliferation and apoptosis induction in Jurkat T-leukemia cells by the new isothiocyanate 4-(methylthio)butylisothiocyanate (MTBITC). To help elucidate whether the effects of MTBITC are specific for cancer cells, we tested MTBITC on freshly isolated, non-transformed human peripheral T lymphocytes. The effects of MTBITC are leukemic-cell-specific and consist of derangements in a critical point of cell-cycle control (G2/M transition). In fact, an increase in the proportion of G2 cells (from about 18% to 50%) was apparent following 24 h of treatment, associated with a decrease in the protein expression of cyclin B1. The expression of cyclin-dependent kinase (CDK) 1 was more mildly attenuated by MTBITC. Our results demonstrated that high concentrations of MTBITC can also induce apoptosis, through an increase of p53 and bax, but not bcl-2, protein expression. No effects of MTBITC were demonstrated on non-transformed T lymphocytes. Taking into account its in vitro antineoplastic activity and selectivity toward leukemia cells, MTBITC can be viewed as a conceptually promising agent in cancer therapy.

Sulforaphane modulates cell cycle and apoptosis in transformed and non-transformed human T lymphocytes.

Isothiocyanates exert chemopreventive effects against chemically induced tumors in animals, modulating enzymes required for carcinogens' activation/detoxification and/or the induction of cell-cycle arrest and apoptosis in tumor cell lines. To investigate the chemopreventive potential of isothiocyanates, we studied proliferation, apoptosis induction and p53, bcl-2 and bax protein expression in Jurkat T-leukemia cells by the isothiocyanate sulforaphane. Sulforaphane caused G(2)/M-phase delay and increase of apoptotic cell fraction in a time- and dose-dependent manner. Necrosis was observed after prolonged exposure to elevated sulforaphane doses. Moreover, it markedly increased p53 and bax protein expression, and slightly affected bcl-2 expression. Since selective targeting and low toxicity for normal host tissues are fundamental requisites for proposed chemopreventive agents such as sulforaphane, we tested sulforaphane on non-transformed phytohemagglutinin-stimulated human T-lymphocytes. We demonstrated that sulforaphane arrested cell-cycle progression in G1 phase by a significant down-modulation of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53, whereas it exerted little effect on bcl-2 and bax levels. These findings indicate that sulforaphane can exert protective effects inhibiting leukemic cell growth. Moreover, sulforaphane is active not only in transformed lymphocytes but also in their normal counterpart. Although in vitro studies do not necessarily predict in vivo outcomes, our findings raise important questions regarding the suitability of sulforaphane for cancer chemoprevention.

Growth inhibition, cell-cycle arrest and apoptosis in human T-cell leukemia by the isothiocyanate sulforaphane.

Glucosinolates (GL) can inhibit, retard or reverse experimental multistage carcinogenesis. When brassica plant tissue is broken, GLs are hydrolyzed by the endogenous enzyme myrosinase (Myr), releasing many products including isothiocyanates (ITC). Synthetic ITCs like sulforaphane exert chemopreventive effects against chemically induced tumors in animals, modulating enzymes required for carcinogens' activation/detoxification and/or the induction of cell-cycle arrest and apoptosis in tumor cell lines. To investigate the chemopreventive potential of ITCs while reproducing the circumstances of dietary contact with sulforaphane, we studied proliferation, apoptosis induction and p53, bcl-2 and bax protein expression in Jurkat T-leukemia cells by sulforaphane, the ITC generated in situ in a quantitative manner by Myr starting from glucoraphanin (GRA). Jurkat cells were treated with different doses of GRA-Myr mixture. Effects on cell growth or survival were evaluated by counting trypan blue-excluding cells. Cell-cycle progression, apoptosis and expression of p53, bax and bcl-2 proteins were analyzed by flow cytometry. Results were analyzed by two-sided Fisher's exact test. Sulforaphane, but not GRA, caused G(2)/M-phase arrest (P = 0.028) and increase of apoptotic cell fraction (P < 0.0001) in a time- and dose-dependent manner. Necrosis was observed after prolonged exposure to elevated sulforaphane doses. Moreover, it markedly increased p53 and bax protein expression, and slightly affected bcl-2 expression. These findings indicate that sulforaphane but not the native GL GRA can exert both protective and toxic effects inhibiting leukemic cell growth. Sulforaphane therefore deserves study as a potential chemopreventive/chemotherapeutic antileukemic agent.

Studies on micronucleus formations in 3 cell lines induced by Tripterygium hypoglaucun Hutch / 第三军医大学学报

Objective　To study the toxicity of Tripterygium Hypolaucun (Level) Hutch (THH). Methods　The flow cytometry technique was used to measure the micronucleus (MN) formation and cell apoptosis of Jurkat, CHE and NIH3T3 cell lines induced by THH. Results　THH could induce micronucleus formation and cell apoptosis of Jurkat, CHE and NIH3T3 cell lines. Quite similar changes were observed in three cell lines, but the Jurkat cells showed more sensitive than others. It was only 25 μl/plate THH that induced the high peak of MN formation at various time points, and only 50 μl/plate that induced the high peak of cell apoptosis. Conclusion　The result indicates that THH has stronger toxicity to Jurkat cells than to CHE and NIH3T3 cells.

Studies on micronucleus formations in 3 cell lines induced by Tripterygium hypoglaucun Hutch / 第三军医大学学报

Objective　To study the toxicity of Tripterygium Hypolaucun (Level) Hutch (THH). Methods　The flow cytometry technique was used to measure the micronucleus (MN) formation and cell apoptosis of Jurkat, CHE and NIH3T3 cell lines induced by THH. Results　THH could induce micronucleus formation and cell apoptosis of Jurkat, CHE and NIH3T3 cell lines. Quite similar changes were observed in three cell lines, but the Jurkat cells showed more sensitive than others. It was only 25 μl/plate THH that induced the high peak of MN formation at various time points, and only 50 μl/plate that induced the high peak of cell apoptosis. Conclusion　The result indicates that THH has stronger toxicity to Jurkat cells than to CHE and NIH3T3 cells.

Studies on micronucleus formations in 3 cell lines induced by Tripterygium hypoglaucun Hutch / 第三军医大学学报

Objective　To study the toxicity of Tripterygium Hypolaucun (Level) Hutch (THH). Methods　The flow cytometry technique was used to measure the micronucleus (MN) formation and cell apoptosis of Jurkat, CHE and NIH3T3 cell lines induced by THH. Results　THH could induce micronucleus formation and cell apoptosis of Jurkat, CHE and NIH3T3 cell lines. Quite similar changes were observed in three cell lines, but the Jurkat cells showed more sensitive than others. It was only 25 μl/plate THH that induced the high peak of MN formation at various time points, and only 50 μl/plate that induced the high peak of cell apoptosis. Conclusion　The result indicates that THH has stronger toxicity to Jurkat cells than to CHE and NIH3T3 cells.