ABSTRACT
The steroidal saponin RCE4 (1ß, 3ß, 5ß, 25S)spirostan1, 3diol 1[αLrhamnopyranosyl(1â2)ßDxylopyranoside], isolated from Reineckia carnea, exerts significant anticervical cancer activity by inducing apoptosis. The potential effect of RCE4 on proliferation inhibition and autophagy induction has rarely been studied. Therefore, the focus of the present study was to investigate the effects of RCE4 on proliferation, and to elucidate the detailed mechanisms involved in autophagy induction in cervical cancer cells. CaSki cells were treated with RCE4 or/and autophagy inhibitors, and the effect of RCE4 on cellular proliferation was assessed by MTT assay. The proautophagic properties of RCE4 were subsequently confirmed using monomeric red fluorescent proteingreen fluorescent proteinmicrotubuleassociated proteins 1A/1B light chain 3B (LC3) adenoviruses and CYTOID autophagy assays, and by assessing the accumulation of lipidmodified LC3 (LC3II). The mechanisms of RCE4induced autophagy were investigated by western blot analysis. The results demonstrated that inhibiting autophagy significantly promoted RCE4induced cell death, indicating that autophagy served a protective role following RCE4 treatment. In addition, RCE4induced autophagy was reflected by increased expression levels of the serine/threonineprotein kinase ULK1, phosphorylated (p)ULK1, pBeclin1 and LC3II, the formation of autophagosomes and autolysosomes, and sequestosome 1 (p62) degradation. Subsequent analysis indicated that RCE4 activated the AMPactivated protein kinase (AMPK) pathway by upregulating AMPK and pAMPK, and also inhibited the PI3K and extracellular signalregulated kinase (ERK) signaling pathways by downregulating pPI3K, pAkt, pmTOR, Ras, cRaf, pcRaf, dual specificity mitogenactivated protein kinase kinase (MEK)1/2, pMEK1/2 and pErk1/2. Additionally, with increased treatment times RCE4 may impair lysosomal cathepsin activity and inhibit autophagy flux by suppressing the expression of AMPK, pAMPK, ULK1, pULK1 and pBeclin1, and upregulating that of p62. These results indicated that the dual RCE4induced inhibition of the PI3K and ERK pathways may result in a more significant antitumor effect and prevent chemoresistance, compared with the inhibition of either single pathway; furthermore, dual blockade of PI3K and ERK, and the AMPK pathway may be involved in the regulation of autophagy caused by RCE4. Taken together, RCE4 induced autophagy to protect cancer cells against apoptosis, but AMPKmediated autophagy was inhibited in the later stages of RCE4 treatment. In addition, autophagy inhibition improved the therapeutic effect of RCE4. These data highlight RCE4 as a potential candidate for cervical cancer treatment.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Asparagaceae/chemistry , Autophagy/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Saponins/pharmacology , Signal Transduction/drug effects , Spirostans/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Cell Line, Tumor , Female , Humans , Saponins/chemistry , Spirostans/chemistry , TOR Serine-Threonine Kinases/metabolism , Uterine Cervical NeoplasmsABSTRACT
<p><b>AIM</b>To propose a new simple and sensitive voltammetric method for determination of proteins.</p><p><b>METHODS</b>Protein with sulfhydryl or disulfide bond in 0.5 mol x L(-1) NaOH, 1.5 x 10(-4) mol x L(-1) Pb2+ and 0.02% tetrabutylammonium iodide was heated in boiling water for 5 minutes. The reactive product gave a well defined reductive adsorption wave at -0.66 V (vs SCE) by means of single sweep polarography, and the height of derivative wave was proportional to the concentration of proteins.</p><p><b>RESULTS</b>The peak height was linearly proportional to bovine serum albumin (BSA) or human serum albumin (HSA) concentration in range of 7.5 x 10(-10) -3.0 x 10(-7) mol x L(-1) (r(BSA) = 0.9995, and r(HSA) = 0.9990). The detection limit of BSA or HSA was 3.0 x 10(-10) mol x L(-1). For lysozyme (Lyso), the concentration range was from 1.4 x 10(-8) to 1.3 x 10(-6) mol x L(-10 (r(Lyso) = 0.9997) and the detection limit was 7.0 x 10(-9) mol x L(-1).</p><p><b>CONCLUSION</b>The method is simple, rapid, sensitive and applicable to the assay of diluted human serum albumin samples.</p>