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ObjectiveTo investigate the effects of hirsutine on proliferation， apoptosis， metastasis， and invasion of human cervical cancer Ca Ski cells and its action mechanism. MethodThe cell proliferation was determined by cell counting kit-8 （CCK-8） assay and the cell apoptosis by Hoechst 33258 staining and flow cytometry. The mRNA expression levels of B cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and p53 were detected by real-time fluorescence quantitative polymerase chain reaction. The scratch test was conducted to detect the cell mobility， followed by the detection of cell invasion ability using a Transwell chamber. The contents of fibronectin （FN）， matrix metalloproteinase-2 （MMP-2）， and matrix metalloproteinase-9 （MMP-9） were measured by enzyme linked immunosorbent assay （ELISA）， and their protein expression levels were assayed by Western blot. ResultCompared with the control group， hirsutine inhibited the proliferation of Ca Ski cells in a dose- and time-dependent manner. The apoptosis rates and Bax and p53 mRNA expression levels in the 8.0， 16.0， 32.0 μmol·L-1 hirsutine groups rose （P<0.05， P<0.01）， while the Bcl-2 mRNA expression and phosphorylated Src （p-Src） and phosphorylated signal transducer and activator of transcription factor 3 （STAT3） protein expression declined （P<0.05， P<0.01）. Compared with the control group， the 1.0， 2.0， 4.0 μmol·L-1 hirsutine groups exhibited lowered cell mobility， number of transmembrane cells， FN， MMP-2 and MMP-9 contents， and hypoxia inducible factor-1α （HIF-1α）， Vimentin， and N-cadherin protein expression （P<0.05， P<0.01）， but elevated E-cadherin protein expression （P<0.01）. ConclusionHirsutine inhibits the proliferation， metastasis， and invasion of human cervical cancer Ca Ski cells and induces their apoptosis， which may be related to its regulation of Src/STAT3 and HIF-1α/epithelial mesenchymal transformation （EMT） signaling pathways.
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AIM:To investigate the effect of hirsutine on hypoxia-induced migration and invasion abilities of human breast cancer MCF-7 cells and its possible mechanism. METHODS:CCK-8 assay was employed to detect the cyto-toxic effect of hirsutine on the MCF-7 cells. Cell migration was observed by wound healing assay,and cell invasion ability was measured by Transwell invasion assay. Western blot was used to analyze the protein levels of hypoxia-inducible factor-1α(HIF-1α),Snail,E-cadherin and matrix metalloproteinase-9 (MMP-9). The mRNA levels of HIF-1α was detected by RT-PCR. RESULTS:Hirsutine remarkably reduced the cell viability from 32 μmol/L(P<0.05),and the IC50value was 62.82 μmol/L. In hypoxia state,MCF-7 cells showed more powerful capabilities of migration and invasion (P<0.05), higher protein levels of HIF-1α,Snail and MMP-9 (P<0.05),lower protein level of E-cadherin(P<0.05),and higher mRNA level of HIF-1α (P<0.05). These hypoxia-induced effects were all inhibited by hirsutine at 16 μmol/L (P<0.05),apart from the mRNA level of HIF-1α. CONCLUSION:Hirsutine inhibits hypoxia-induced migration and inva-sion in human breast cancer MCF-7 cells most likely via down-regulation of the protein levels of HIF-1α,Snail and MMP-9,and up-regulation of the protein level of E-cadherin.
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The crude Chinese drug “Uncaria hook” is a hook, or a twig with an attached hook of Uncaria plants seen in today's Chinese and Japanese medicinal markets. However, through herbological studies we found that the botanical origin of Uncaria hook was Uncaria rhynchophylla (Miq.) Miq., and that until the middle of Ming Dynasty in ancient China, the medicinal part used was the twig bark, not the hook itself, and use of the twig with hook was begun in the later Ming Dynasty. This change in practice seems to have been influenced by herbal descriptions written in the Ming Dynasty. Some of these stated that the medical effect of hooks was stronger than that of the bark.To determine the appropriate medicinal part of this crude drug in terms of chemical quality, we analyzed the alkaloid contents of Uncaria rhynchophylla bark, hooks, and twigs collected in Japan. Our result showed that the alkaloid content of the bark was higher than that of the twigs and hooks. Rhynchophylline and hirsutine, the alkaloid contained in Uncaria hook, were reported to improve memory learning and to cure hypertension, respectively. Since the alkaloid content profile of the bark was different from that of the hook, a question arose as to whether the medicinal properties of the part commonly used as “Uncaria hook” meet the requirement of the crude drug. Further pharmacological study is expected.