Ronit Rotem

Jasmonates: novel anticancer agents acting directly and selectively on human cancer cell mitochondria.

We reported previously that jasmonates can kill human cancer cells. Many chemotherapeutic drugs induce mitochondrial membrane permeability transition, membrane depolarization, osmotic swelling, and release of cytochrome c, involving the opening of the permeability transition pore complex (PTPC). Because jasmonates exert their cytotoxic effects independent of transcription, translation, and p53 expression, we hypothesized that these compounds may act directly on mitochondria. Mitochondrial membrane depolarization was determined by flow cytometry, and cytochrome c release by Western blotting. Mitochondria were isolated by mechanical lysis and differential centrifugation. Cytotoxicity was measured by a tetrazolium-based assay, and mitochondrial swelling by spectrophotometry. Jasmonates induced membrane depolarization and cytochrome c release in intact human cancer cell lines. Jasmonates induced swelling in mitochondria isolated from Hep 3B hepatoma cells, but not in mitochondria isolated from 3T3 nontransformed cells or from normal lymphocytes, in a PTPC-mediated manner. Methyl jasmonate induced the release of cytochrome c from mitochondria isolated from cancer cell lines in a PTPC-mediated manner, but not from mitochondria isolated from normal lymphocytes. A correlation was found between cytotoxicity of methyl jasmonate and the percentage of leukemic cells in the blood of patients with chronic lymphocytic leukemia (CLL). Jasmonates induced membrane depolarization in CLL cells, and swelling and release of cytochrome c in mitochondria isolated from these cells. In conclusion, jasmonates act directly on mitochondria derived from cancer cells in a PTPC-mediated manner, and could therefore bypass premitochondrial apoptotic blocks. Jasmonates are promising candidates for the treatment of CLL and other types of cancer.

Identification of Extracellular Signal-regulated Kinase 1/2 and p38 MAPK as Regulators of Human Sperm Motility and Acrosome Reaction and as Predictors of Poor Spermatozoan Quality

Mature spermatozoa acquire progressive motility only after ejaculation. Their journey in the female reproductive tract also includes suppression of progressive motility, reactivation, capacitation, and hyperactivation of motility (whiplash), the mechanisms of which are obscure. MAPKs are key regulatory enzymes in cell signaling, participating in diverse cellular functions such as growth, differentiation, stress, and apoptosis. Here we report that ERK1/2 and p38 MAPK are primarily localized to the tail of mature human spermatozoa. Surprisingly, c-Jun N-terminal kinase 1/2, which is thought to be ubiquitously expressed, could not be detected in mature human spermatozoa. ERK1/2 stimulation is downstream to protein kinase C (PKC) activation, which is also present in the human sperm tail (PKCβI and PKCϵ). ERK1/2 stimulates and p38 inhibits forward and hyperactivated motility, respectively. Both ERK1/2 and p38 MAPK are involved in the acrosome reaction. Using a proteomic approach, we identified ARHGAP6, a RhoGAP, as an ERK substrate in PMA-stimulated human spermatozoa. Inverse correlation was obtained between the relative expression level of ERK1 or the relative activation level of p38 and sperm motility, forward progression motility, sperm morphology, and viability. Therefore, increased expression of ERK1 and activated p38 can predict poor human sperm quality.

Jasmonates induce nonapoptotic death in high-resistance mutant p53-expressing B-lymphoma cells

Mutations in p53, a tumor suppressor gene, occur in more than half of human cancers. Therefore, we tested the hypothesis that jasmonates (novel anticancer agents) can induce death in mutated p53‐expressing cells. Two clones of B‐lymphoma cells were studied, one expressing wild‐type (wt) p53 and the other expressing mutated p53. Jasmonic acid and methyl jasmonate (0.25–3 mM) were each equally cytotoxic to both clones, whereas mutant p53‐expressing cells were resistant to treatment with the radiomimetic agent neocarzinostatin and the chemotherapeutic agent bleomycin. Neocarzinostatin and bleomycin induced an elevation in the p53 levels in wt p53‐expressing cells, whereas methyl jasmonate did not. Methyl jasmonate induced mostly apoptotic death in the wt p53‐expressing cells, while no signs of early apoptosis were detected in mutant p53‐expressing cells. In contrast, neocarzinostatin and bleomycin induced death only in wt p53‐expressing cells, in an apoptotic mode. Methyl jasmonate induced a rapid depletion of ATP in both clones. In both clones, oligomycin (a mitochondrial ATP synthase inhibitor) did not increase ATP depletion induced by methyl jasmonate, whereas inhibition of glycolysis with 2‐deoxyglucose did. High glucose levels protected both clones from methyl jasmonate‐induced ATP depletion (and reduced methyl jasmonate‐induced cytotoxicity), whereas high levels of pyruvate did not. These results suggest that methyl jasmonate induces ATP depletion mostly by compromising oxidative phosphorylation in the mitochondria. In conclusion, jasmonates can circumvent the resistance of mutant p53‐expressing cells towards chemotherapy by inducing a nonapoptotic cell death.

Contrasting effects of aspirin on prostate cancer cells : Suppression of proliferation and induction of drug resistance

Aspirin is widely used as a preventive measure against occlusive vascular diseases. Since the age group in which aspirin use has become prevalent is similar to the one presenting with prostate cancer, we decided to examine the potential effects of aspirin on prostate cancer.

Protein kinase C ε mediates the induction of P-glycoprotein in LNCaP prostate carcinoma cells

Abstract P-glycoprotein (P-gp) mediates drug resistance. Protein kinase C (PKC) expression correlates with drug resistance in several types of cancer. We determined whether PKC signals the induction of P-gp in LNCaP human prostate cancer cells, and identified a specific isozyme involved, in a model of aspirin-induced P-glycoprotein expression. An inhibitor of PKC activity, and a specific peptide inhibitor of PKC e translocation, suppressed the induction of P-gp. The PKC activator ingenol, but not OAG, induced P-gp expression in a dose-dependent manner. Based on our results, we conclude that PKC e mediates the induction of P-gp. Accordingly, PKC e is activated and translocates from the membrane fraction to the cytoskeleton fraction in aspirin-treated cells. The findings of this study point to PKC e as a signalling molecule for the induction of P-gp in LNCaP prostate cancer cells.

Stress-responsive JNK mitogen-activated protein kinase mediates aspirin-induced suppression of B16 melanoma cellular proliferation

Available anticancer drugs do not seem to modify the prognosis of metastatic melanoma. Salicylate and acetyl salicylic acid (aspirin) were found to suppress growth in a number of transformed cells, that is, prostate and colon. Therefore, we studied the direct effects of aspirin on metastatic B16 melanoma cells. Aspirin at a plasma‐attainable and nontoxic level suppressed the proliferation of B16 cells. Aspirin induced the activation of p38 and c‐Jun N‐terminal kinase (JNK) mitogen‐activated protein kinases. Inhibition of JNK, but not p38, decreased the suppressive effect of aspirin upon the proliferation of B16 cells. The aspirin‐induced reduction in B16 proliferation was cumulative over time. Aspirin and the chemotherapeutic drug 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (BCNU) induced B16 cell death synergistically. In addition to the murine B16 cell line, the proliferation of SK‐28 human melanoma cells was also suppressed by aspirin. In conclusion, aspirin suppresses the proliferation of metastatic B16 cells in a JNK‐dependent mechanism.

The anticancer agent methyl jasmonate induces activation of stress-regulated c-Jun N-terminal kinase and p38 protein kinase in human lymphoid cells.

We have recently discovered that plant stress hormones, that regulate cell death in stressed plants, are capable of suppressing the proliferation and inducing apoptosis in various human transformed cells including leukemia, melanoma, prostate and breast cancer cells. That study focused on several plant stress hormones, that is, salicylic acid and jasmonates. Methyl jasmonate (MJ) was determined to be the most potent agent. Furthermore, while MJ killed Molt-4 human lymphoblastic leukemia cells in an efficient manner, it did not affect normal human peripheral blood lymphocytes. Finally, MJ administered in vivo significantly increased the survival of lymphoma-bearing mice. c-Jun N-terminal kinase (JNK) and p38 are groups of mammalian mitogen-activated protein kinases (MAPK) that can be induced by cellular stress. JNK and p38 influence transcription activator protein 1 (AP-1) activity by both increasing the abundance of AP-1 forming proteins, and by stimulating their activity directly. AP-1 regulates the expression of various stress-related genes, including genes that mediate cellular injury (eg, IL-1b, IL-8, iNOS, etc). Stresses that induce JNK, p38 and AP-1 activities are numerous, for example, UV radiation, oxidative stress, heat shock, osmotic shock, cell stretching, pathogens, heavy metals and anticancer drugs such as taxol. The signaling pathways involving JNK, p38 and AP-1 mediate the death resulting from exposure to various stressors. Plant stress responses involve induction of cell death, and have recently been shown to upregulate MAPK and upstream enzymes, MAPK kinase kinase, in plant cells. Therefore, we hypothesized that analogous to the induction of stress signaling in plant cells, the plant stress hormone MJ also induces stress signaling in human cells. To test this hypothesis, we analyzed stress-regulated MAPK signaling events in MJ-treated human Molt-4 leukemic cells and normal peripheral blood lymphocytes, and assessed the role of these signaling steps in MJinduced cytotoxicity. Molt-4 human lymphoblastic leukemia cells were exposed to the plant stress hormone MJ and the phosphorylation state of p38 and c-Jun was determined by Western blotting. c-Jun phosphorylation reflects the activity of the JNK MAPK, while p38 phosphorylation reflects its activation by upstream kinases. We administered MJ at 3 mM. This concentration was chosen based on our previous results showing that MJ at 3 mM induces strong cytotoxicity towards Molt-4 cells. In addition, MJ is a plant stress hormone and we chose its concentration based on the plasma concentrations achieved upon administration of a well-studied plant stress hormone, salicylic acid. Salicylic acid is a nonsteroidal anti-inflammatory drug. While most nonsteroidal anti-inflammatory drugs (such as indomethacin) act in the micromolar range, salicylates act in the low millimolar range. The highest nontoxic pharmacological concentration of salicylate used in humans is approximately 3 mM. MJ induced time-dependent c-Jun phosphorylation that was inhibited by SB202190, a JNK inhibitor (Figure 1, upper panels). Similarly, MJ induced the phosphorylation of p38, again in a timedependent manner (Figure 1, lower panels). Both signaling events were already detectable 10 min following exposure to MJ. The levels of nonphosphorylated c-Jun and p38 were practically unchanged throughout the experiment (Figure 1). We further analyzed MJ-induced stress signaling by determining AP-1 activity in Molt-4 cells. To that end, we constructed a reporter plasmid expressing ECFP (a fluorescent protein) under the control of AP-1 (by inserting an AP-1 DNAbinding sequence: 50-GAAATTCCAGAGAGTCATCAGAAGA-30), and transfected Molt-4 cells with that plasmid. Fluorescence of these cells (Molt-4-AT) reflects AP-1-dependent ECFP gene transcription and translation. Molt-4-AT cells were treated with or without MJ or the classical AP-1 inducer TPA (as a positive control). Fluorescence microscopy revealed that while untreated cells did not exhibit fluorescence, both TPAand MJ-treated cells were brightly fluorescent (Figure 2a). Clearly, this was an indication that MJ induced AP-1 activity in human Molt-4 leukemic cells. In addition, we evaluated induction of AP-1 activity upon exposure to MJ at different concentrations, for various time periods, using flow cytometry as shown in Figure 2b. The results show that MJ induced AP-1 activity in Molt-4-AT cells doseand time-dependently. For example, only the highest concentration of MJ used (3 mM) induced some AP-1 activity within 2 h, while MJ at 0.5 mM was sufficient to induce AP-1 activity within 6 h. Thus, MJ induced JNK and p38 activities as well as the activity of AP-1, a transcription factor known to be regulated by these MAPK. In order to establish a cause-and-effect relationship between induction of MAPK and of AP-1 activities in MJ-treated cells, we employed inhibitors of JNK and p38. To that end, we determined AP-1 activity in MJtreated Molt-4-AT cells in the presence or absence of SB203580 (a specific p38a and p38b inhibitor that does not inhibit the activity of JNK or of any other MAPK), or SB202190 (an agent that inhibits JNK as well as p38a and p38b). While SB202190 exhibited a strong inhibitory effect on AP-1 induction by MJ, SB203580 had no effect at all (Figure 2c). Moreover, while SB202190 was employed at 7.5 mM, we raised the concentration of SB203580 up to the maximal nontoxic level (25mM) and still did not detect any inhibitory effect of the latter on the induction of AP-1 by MJ. Since a specific inhibitor of p38a and p38b (SB203580) was ineffective, whereas an inhibitor of p38a, p38b as well as JNK (SB202190) did suppress the induction of AP-1, we concluded that JNK mediates the induction of AP-1 activity by MJ. MJ is selectively cytotoxic towards transformed cells while sparing normal lymphocytes. Therefore, we decided to investigate whether the effect of MJ on p38 and JNK activities in normal lymphocytes differs from its effect on these enzymes in leukemic cells. To that end, we exposed peripheral blood lymphocytes from healthy individuals to MJ at 3 mM and measured the phosphorylation of c-Jun and p38. MJ Leukemia (2003) 17, 2230–2256 & 2003 Nature Publishing Group All rights reserved 0887-6924/03

Clomiphene citrate treatment in oligozoospermia: comparison between two regimens of low-dose treatment.

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Determination of Urinary Luteinizing Hormone for Prediction of Ovulation

Clomiphene citrate (CC) is a well-known drug in fertility clinics that is used for increasing gonadotropin secretion. The present study was planned in order to evaluate the efficiency of a new regimen of treatment by 25 mg on alternate days (group A), compared to a daily dose of 25 mg (25 days on, 5 days off, group B), for 4 months. Semen quality was assessed in two matched groups, which consisted of 45 and 44 normogonadotropic oligoterato-asthenozoospermic (OTA) men, respectively. Nine men in group A and 22 in group B did not respond to therapy by improvement in semen quality. The statistical evaluation of the results revealed group A to yield the highest improvement in sperm concentration (P less than 0.0008) and total sperm count (P less than 0.004). Sperm motility was improved only in group A. No changes were recorded in the morphology of the sperm cells or in semen volume. Pregnancy rate after 6 months of follow-up was 26.7% and 20.5%, in couples of groups A and B, respectively. This study implicates the use of CC (25 mg on alternate days) in andrologic clinics as one of the recommended drugs for normogonadotropic OTA subfertile men in order to achieve a significant increase in sperm concentration and total sperm count.

The use of an electric freezer in human semen banking

Detection of the approximate time of ovulation is important in the in vitro fertilization-embryo transfer programs in order to avoid a possible early surge of luteinizing hormone (LH) before human chorionic gonadotropin administration in the induction of ovulation and for timing of artificial insemination. In the present study correlation between the detection of LH in urine and serum levels (mean +/- SEM) of estradiol greater than 557 +/- 118 pg/ml, progesterone less than 1.9 +/- 0.37 ng/ml and leading follicle diameter greater than 26.2 +/- 1.3 mm (mean +/- SEM) was found. Thus, the prediction of ovulation via detection of urinary LH in urine by a dipstick procedure is a good semiquantitative method, accurate enough, reliable, cheap and acceptable by the women.