Studies with 15α-methoxypuupehenol (15α-MP), obtained from the extracts of Hyrtios sp., identified putative targets that are associated with its antitumor effects against human glioblastoma (GBM) and breast cancer. In the human GBM (U251MG) or breast cancer (MDA-MB-231) cells, treatment with 15α-MP repressed pY705Stat3 (Signal Transducer and Activator of Transcription 3), pErk1/2 (extracellular signal-regulated kinase), pS147CyclinB1, pY507Alk (Anaplastic lymphoma kinase), and pY478ezrin levels, and induced pS10merlin, without inhibiting pJAK2 (Janus kinase) or pAkt induction. 15α-MP treatment induced loss of viability of breast cancer (MDA-MB-231, MDA-MB-468) and GBM (U251MG) lines and GBM patient-derived xenograft cells (G22) that harbor aberrantly-active Stat3, with only moderate or little effect on the human breast cancer, MCF7, colorectal adenocarcinoma Caco-2, normal human lung fibroblast, WI-38, or normal mouse embryonic fibroblast (MEF Stat3fl/fl) lines that do not harbor constitutively-active Stat3, or the Stat3 null (Stat3−/−) mouse astrocytes. 15α-MP-treated U251MG cells have severely impaired F-actin organization and altered morphology, including the cells rounding up, and undergo apoptosis, compared to a moderate, reversible morphology change observed for similarly-treated mouse astrocytes. Treatment further inhibited U251MG or MDA-MB-231 cell proliferation, anchorage-independent growth, colony formation, and migration in vitro, while only moderately or weakly affecting MCF7 cells or normal mouse astrocytes. Oral gavage delivery of 15α-MP inhibited the growth of U251MG subcutaneous tumor xenografts in mice, associated with apoptosis in the treated tumor tissues. Results together suggest the modulation of Stat3, CyclinB1, Alk, ezrin, merlin, and Erk1/2 functions contributes to the antitumor effects of 15α-MP against GBM and breast cancer progression.

Glioblastoma multiforme (GBM) is a malignant brain tumor that lacks effective therapeutics and has a high mortality rate. Constitutively-activated signal transducer and activator of transcription 3 (STAT3) protein is prevalent and implicated in GBM phenotype and represents a valid anti-GBM target. To discover novel STAT3 inhibitors suitable for GBM therapy, natural product extracts were investigated. Whole extracts from marine organisms were first screened for effects against cell viability using the human glioma model line, U251MG that harbors aberrantly-active STAT3 and normal fibroblasts that do not. The screening campaign identified the puupehenol, CC-1-2 from the whole extracts of leiodermatium sp as a bioactive compound that suppressed U251MG cell viability, with an IC50 4.1±0.1 µM, compared to an IC50 value of 7.3±0.3 µM against normal NIH3T3 mouse fibroblasts. Western blot analysis showed pSTAT3 levels in U251MG cells substantially decreased in a time-dependent manner in response to CC-1-2. Decreased pSTAT3 in treated cells occurred in parallel with partial suppression of both phospho- Janus kinase (Jak) 2 and pSrc levels and no induction of protein tyrosine phosphatase, PTP1B. Phosphorylated extracellular signal-regulated kinase (Erk) 1/2 levels also decreased in time-dependent manner, while pAkt remained unchanged in U251MG cells under the same treatment conditions. Furthermore, treatment of U251MG cells with CC-1-2 led to dramatic morphological changes that occurred in a dose- and time-dependent manner, compared to minimal changes observed for similarly-treated MCF-7 breast cancer cells that do not harbor aberrantly-active STAT3. Colony survival assay of U251MG cells revealed single treatment with 1-10 µM CC-1-2 decreased colony sizes and numbers in a dose-dependent manner, compared to moderate effects on MCF-7. In vitro wound-healing assay further showed reduced U251MG cell migration into the denuded area in response to CC-1-2 treatment. CC-1-2 further induced apoptosis in U251MG cells in a dose-dependent manner and suppressed the expression of c-Myc, Cyclin D1, and Bcl-2 levels, which are known STAT3-regulated genes. Studies together identify CC-1-2 as a natural product inhibitor of STAT3 that preferentially induces antitumor cell effects against human glioblastoma cells. Citation Format: Tyvette S. Hilliard, Christopher Chock, Philip Williams, James Turkson. Puupehenol natural product inhibits STAT3 signaling and induces antitumor cell effects in vitro against human glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1783. doi:10.1158/1538-7445.AM2014-1783