Cucurbitacins are a group of diverse triterpenoid substances isolated from plants with medicinal properties. One particularly potent family member is cucurbitacin B (CuB). The antiproliferative effects of CuB against human breast cancer cells were tested. Six human breast cancer cell lines were examined because they represent a diverse mix of breast cancer subtypes varying in expression of estrogen receptor (ER), Her2/neu, and p53 mutation. The antiproliferative effect of CuB were also studied in vivo. The effective dose inhibiting 50% growth (ED 50 ) was between 10 -8 M and 10 -7 M for this collection of breast cancer cell lines. These cells underwent rapid morphologic changes after 15-20 min exposure to CuB (5´10 -7 M), which was associated with disruption of the microtubules and F-actin, as observed by confocal microscopy. C ucurbitacins were originally isolated from Cucurbitaceae plants. They are a group of diverse triterpenoid molecules containing a cucurbitane skeleton characterized by a 19-(10→9β)-abeo-10α-lanost-5-ene (Fig. 1).(1) They have been reported to possess a broad range of biological effects such as chemoprevention and hepatoprotection, as well as anti-inflammatory, antimicrobial and antitumor activities.(2-10) Cucurbitacins have been reported to inhibit several types of cancers including those originating in the prostate, (9) lung and breast (11) as well as choriocarcinoma.(12) Among the various cucurbitacins, the most abundant is cucurbitacin B (CuB) and its analog dihydrocucurbitacin B.(13) Our preliminary data suggest that CuB is one of the most potent compounds in this family.Breast cancer afflicts about one in nine women, with approximately 43 000 deaths per year in the USA from this disease. Although great progress has been made in both controlling the disease and preventing recurrence, additional novel therapies are needed leading us to focus on investigating the effects of CuB on breast cancer in vitro and in vivo. Materials and Methods Cells and compounds. MCF-7, MDA-MB-231, MDA-MB-453, T47D, BT474, SK-BR-3 and ZR-75-1 cells (American TypeCulture Collection, Manassas, VA, USA) were maintained at 37°C in 5% CO 2 in Roswell Park Memorial Institute media (RPMI)-1640 (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Invitrogen), 10 U/mL penicillin and 10 mg/ mL streptomycin (Invitrogen). CuB was kindly provided by CK Life Sciences International Inc., Hong Kong.Cytoskeletal staining for b-tubulin and F-actin. After incubation with CuB, culture media were removed and cells were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS, and incubated with fluorescein isothyanate (FITC)-conjugated antiβ-tubulin antibody (Sigma Chemical, St. Louis, MO, USA) and/or rhodaminephalloidin (Invitrogen) to detect filamentous (F)-actin. Confocal images were collected on a Leica microscope (Leica Microsystems, Wetzlar, Germany).Animal experiments. MDA-MB-231 (1 × 10 6 ) breast cancer cells were resuspended in 100-μL matrigel and were inocul...

Glioblastoma Multiforme (GBM) is almost inevitably a fatal tumor of the brain with most individuals dying within 1 year of diagnosis. It is the most frequent brain tumor in adults. Dose-response studies showed that Cucurbitacin B inhibited 50% growth (ED 50 ) of 5 human GBM cell lines in liquid culture at 10 27 M. Soft-gel assays demonstrated that nearly all of the GBM clonogenic cells were inhibited at 10 28 M of Cucurbitacin B. FACS analysis found that the compound (10 27 M, 24 hr) caused G2/M arrest. The GBM cells underwent profound morphologic changes within 15-30 min after exposure to Cucurbitacin B (10 27 M), rounding up and losing their pseudopodia associated with disruption of actin and microtubules, as observed by immunoflourescence. Cucurbitacin B (10 27 M) caused prominent multinucleation of the cells after they were pulse-exposed (48 hr) to the drug, washed and cultured in normal medium for an additional 2 days. The drug (10 27 M, 3-24 hr) increased levels of p-p38, p-JNK and p-JUN in U87 and T98G GBM cell lines as seen by Western blot. Interestingly, alterations in cell morphology caused by Cucurbitacin B (10 27 M) were blocked by the JNK inhibitor SP600125. In summary, Cucurbitacin B has a prominent anti-proliferative activity on GBM cells; and at least in part, the mode of action is by affecting the cytoskeleton, as well as, the JNK pathway. Clinical trails of this drug should be pursued in GBM.

The JAK2 mutation JAK2V617F is found frequently in patients with myeloproliferative disorders (MPD) and transforms hematopoietic cells to cytokine-independent proliferation when expressed with specific cytokine receptors. The Src homology 2 (SH2) and pleckstrin homology (PH) domain-containing adaptor protein Lnk (SH2B3) is a negative regulator of hematopoietic cytokine signaling. Here, we show that Lnk is a potent inhibitor of JAK2V617F constitutive activity. Lnk down-regulates JAK2V617F-mediated signaling and transformation in hematopoietic Ba/F3-erythropoietin receptor cells. Furthermore, in CFU assays, Lnk-deficient murine bone marrow cells are significantly more sensitive to transformation by JAK2V617F than wild-type (WT) cells. Lnk, through its SH2 and PH domains, interacts with WT and mutant JAK2 and is phosphorylated by constitutively activated JAK2V617F. Finally, we found that Lnk levels are high in CD34(+) hematopoietic progenitors from MPD patients and that Lnk expression is induced following JAK2 activation. Our data suggest that JAK2V617F is susceptible to endogenous negative-feedback regulation, providing new insights into the molecular pathogenesis of MPD.