Cucurbitacin I suppressed stem-like property and enhanced radiation-induced apoptosis in head and neck squamous carcinoma--derived CD44(+)ALDH1(+) cells.

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer worldwide. Signal transducers and activators of transcription 3 (STAT3) signaling is reported to promote tumor malignancy and recurrence in HNSCC. Cucurbitacins, triterpenoid derivatives, are strong STAT3 inhibitors with anticancer properties. Recent studies have shown aldehyde dehydrogenase 1 (ALDH1) to be a marker of cancer stem cells (CSC) in HNSCC. The aim of this study was to investigate the therapeutic effect of cucurbitacin I in HNSCC-derived CSCs. Using immunohistochemical analysis, we firstly showed that CD44, ALDH1, and phosphorylated STAT3 (p-STAT3) were higher in high-grade HNSCCs, and that triple positivity for CD44/ALDH1/p-STAT3 indicated a worse prognosis for HNSCC patients. Secondly, CD44(+)ALDH1(+) cells isolated from seven HNSCC patients showed greater tumorigenicity, radioresistance, and high expression of stemness (Bmi-1/Oct-4/Nanog) and epithelial-mesenchymal-transitional (Snail/Twist) genes as p-STAT3 level increased. Furthermore, we found that cucurbitacin I (JSI-124) can effectively inhibit the expression of p-STAT3 and capacities for tumorigenicity, sphere formation, and radioresistance in HNSCC-CD44(+)ALDH1(+). Notably, 150 nmol/L cucurbitacin I effectively blocked STAT3 signaling and downstream survivin and Bcl-2 expression, and it induced apoptosis in HNSCC-CD44(+)ALDH1(+). Moreover, microarray data indicated that 100 nmol/L cucurbitacin I facilitated CD44(+)ALDH1(+) cells to differentiate into CD44⁻ALDH1⁻ and enhanced the radiosensitivity of HNSCC-CD44(+)ALDH1(+). Xenotransplant experiments revealed that cucurbitacin I combined with radiotherapy significantly suppressed tumorigenesis and lung metastasis and further improved the survival rate in HNSCC-CD44(+)ALDH1(+)-transplanted immunocompromised mice. Taken together, our data show that cucurbitacin I, STAT3 inhibitor, reduces radioresistant, distant-metastatic, and CSC-like properties of HNSCC-CD44(+)ALDH1(+) cells. The potential of cucurbitacin I as a radiosensitizer should be verified in future anti-CSC therapy.

Synergistic effect of endothelin-1 and cyclic AMP on glucose transport in 3T3-L1 adipocytes.

We have demonstrated previously that chronic exposure to endothlin-1 enhances glucose transport in 3T3-L1 adipocytes via augmented GLUT1 mRNA and protein accumulation. In the present study, we further examined the combined effect of endothelin-1 (ET-1) and cAMP on glucose transport. In cells pretreated with ET-1 and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. Immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both ET-1 and 8-bromo cAMP, greater than the additive effect of each agent alone. Further examination demonstrated that the stability of GLUT1 mRNA was markedly enhanced in the presence of both ET-1 and cAMP. To investigate the transcriptional activation of Glut1 gene, transient transfection of cells with luciferase reporter construct driven by Glut1 promoter was performed. We found that Glut1 transcription was also increased by ET-1 and cAMP in a synergistic fashion. In addition, similar synergy between ET-1 and beta-adrenergic agonists on glucose transport was found. The synergistic action of ET-1 with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor, and was mimicked by 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA), a PKC activator. Furthermore, PMA was found to act synergistically with 8-bromo cAMP to induce Glut1 transcription and ET-1 was shown to activate novel PKCdelta and PKC. Taken together, these results indicate that ET-1 may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression via a PKC-dependent mechanism.