The anticancer phytochemical rocaglamide inhibits Rho GTPase activity and cancer cell migration

Chemotherapy is one of the pillars of anti-cancer therapy. Although chemotherapeutics cause regression of the primary tumor, many chemotherapeutics are often shown to induce or accelerate metastasis formation. Moreover, metastatic tumors are largely resistant against chemotherapy. As more than 90% of cancer patients die due to metastases and not due to primary tumor formation, novel drugs are needed to overcome these shortcomings. In this study, we identified the anticancer phytochemical Rocaglamide (Roc-A) to be an inhibitor of cancer cell migration, a crucial event in metastasis formation. We show that Roc-A inhibits cellular migration and invasion independently of its anti-proliferative and cytotoxic effects in different types of human cancer cells. Mechanistically, Roc-A treatment induces F-actin-based morphological changes in membrane protrusions. Further investigation of the molecular mechanisms revealed that Roc-A inhibits the activities of the small GTPases RhoA, Rac1 and Cdc42, the master regulators of cellular migration. Taken together, our results provide evidence that Roc-A may be a lead candidate for a new class of anticancer drugs that inhibit metastasis formation

Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution

Artesunate induces ROS-mediated apoptosis in doxorubicin-resistant T leukemia cells.

BACKGROUND: A major obstacle for successful cancer treatment often is the development of drug resistance in cancer cells during chemotherapy. Therefore, there is an urgent need for novel drugs with improved efficacy against tumor cells and with less toxicity on normal cells. Artesunate (ART), a powerful anti-malarial herbal compound, has been shown to inhibit growth of various tumor cell lines in vitro and of xenografted Kaposi's sarcoma in mice in vivo. However, the molecular mechanisms by which ART exerts its cytotoxicity have not been elucidated. The ART-class of anti-malarial compounds is attractive due to their activity against multidrug-resistant Plasmodium falciparum and Plasmodium vivax strains. Another salient feature of these compounds is the lack of severe side effects in malaria patients. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we used T-cell leukemias as a model system to study the molecular mechanisms of ART-induced apoptosis. The most typical anticancer drugs are DNA intercalators such as Doxorubicin. To investigate drug sensitivity and resistance, we chose a Doxorubicin-resistant leukemia cell line and investigated the killing effect of ART on these cells. We show that ART induces apoptosis in leukemic T cells mainly through the mitochondrial pathway via generation of reactive oxygen species (ROS), a mechanism different from Doxorubicin. This is confirmed by the fact that the antioxidant N-Acetyle-Cysteine (NAC) could completely block ROS generation and, consequently, inhibited ART-induced apoptosis. Therefore, ART can overcome the Doxorubicin-resistance and induce the Doxorubicin-resistant leukemia cells to undergo apoptosis. We also show that ART can synergize with Doxorubicin to enhance apoptotic cell death in leukemic T cells. This synergistic effect can be largely explained by the fact that ART and Doxorubicin use different killing mechanisms. CONCLUSIONS: Our studies raise the possibility to develop ART in combination with other established anticancer drugs which induce apoptosis through the pathways or mechanisms different from ART

ART sensitizes malignant T cells to Doxorubicin-induced apoptosis.

<p>Jurkat (A) and CEM (B) cells were treated with combinations of different doses of ART and Doxorubicin for 24 h as indicated. The drugs were added at the same time. Apoptotic cell death was quantified by DNA fragmentation in triplicates. Results are representative of three independent experiments. The p value was determined by a two-way ANOVA test with factors Dox-, ART-, and their interaction. For Jurkat cells, the p values are p<0.0001 for Dox- and ART-treatment and p = 0.0006 for the combination treatment. For CEM cells, the p values are p<0.0001 for Dox-, ART- as well as the combination treatment.</p

ART induces Doxorubicin-resistant leukemic cells to undergo apoptosis by a mechanism different from the one induced by Doxorubicin.

<p>(A) ART induces apoptosis in Doxorubicin-resistant leukemic cells. Doxorubicin-resistant (CEM-Dox_R) and parental (CEM-parental) CEM cells were treated with either 1 µg/ml of ART or 0.5 µg/ml of Doxorubicin for 24 h. Apoptosis was determined by FSC/SSC (left panel) and DNA fragmentation (right panel). Results are representative of two independent experiments. (B) NAC inhibits ART-induced apoptosis in Doxorubicin-resistant leukemic cells. CEM-Dox_R cells were treated with different doses of ART in the presence or absence of NAC (15 mM). Apoptotic cell death was determined by DNA fragmentation in triplicates. (C) and (D) NAC does not inhibit Doxorubicin-induced apoptosis. CEM-parental (C) and Jurkat (D) cells were treated either with ART or Doxorubicin in the presence or absence of NAC. Apoptotic cell death was determined by DNA fragmentation. Results are representative of four independent experiments. The p value was determined by the statistic program of Microsoft Excel. (E) ART induces ROS generation in CEM-Dox_R cells. CEM-Dox_R cells were treated with 4 µg/ml of ART in the presence or absence of NAC (15 mM) for 30 min. The redox status was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000693#pone-0000693-g003" target="_blank">figure 3</a>. (F) Doxorubicin does not induce ROS in leukemic T cells. CEM-Dox_R, CEM-parental and Jurkat cells were treated with 0.5 µg/ml of Doxorubicin in the presence or absence of NAC for 30 min. The redox status was measured as in (E). Results are representative of three independent experiments. (G) Doxorubicin does not interfere with the redox assay. CEM cells were treated with Doxorubicin (2 µg/ml) or ART (4 µg/ml) alone or in combination for 30 min. The redox status was measured as in F.</p

ROS mediate ART-induced apoptosis in leukemia cells.

<p>(A) Jurkat cells were treated with 4 µg/ml of ART for different times as indicated and the redox status was monitored by the oxidation-sensitive fluorescent dyes for H₂O₂. (B) CEM cells were treated with 0.5 µg/ml doses ART. After 30 min, the redox status was measured as in (A). (C) and (D) ART-induced apoptosis was blocked by the antioxidant NAC. Jurkat (C) and CEM (D) cells were treated with ART in the presence (15 mM) or absence of NAC for 24 h. Apoptotic cell death was analyzed by FSC/SSC in triplicates. Results are representative of four independent experiments.</p