RCE-4, a potential anti-cervical cancer drug isolated from Reineckia carnea, induces autophagy via the dual blockade of PI3K and ERK pathways in cervical cancer CaSki cells

The steroidal saponin RCE-4 (1 beta, 3 beta, 5 beta, 25S)-spirostan-1, 3-diol 1-[alpha -L-rhamnopyranosyl-(1 -> 2)-beta -D-xylopyranoside], isolated from Reineckia carnea, exerts significant anti-cervical cancer activity by inducing apoptosis. The potential effect of RCE-4 on proliferation inhibition and autophagy induction has rarely been studied. Therefore, the focus of the present study was to investigate the effects of RCE-4 on proliferation, and to elucidate the detailed mechanisms involved in autophagy induction in cervical cancer cells. CaSki cells were treated with RCE-4 or/and autophagy inhibitors, and the effect of RCE-4 on cellular proliferation was assessed by MTT assay. The pro-autophagic properties of RCE-4 were subsequently confirmed using monomeric red fluorescent protein-green fluorescent protein-microtubule-associated proteins 1A/1B light chain 3B (LC3) adenoviruses and CYTO-ID autophagy assays, and by assessing the accumulation of lipid-modified LC3 (LC3II). The mechanisms of RCE-4-induced autophagy were investigated by western blot analysis. The results demonstrated that inhibiting autophagy significantly promoted RCE-4-induced cell death, indicating that autophagy served a protective role following RCE-4 treatment. In addition, RCE-4-induced autophagy was reflected by increased expression levels of the serine/threonine-protein kinase ULK1, phosphorylated (p)-ULK1, p-Beclin-1 and LC3II, the formation of autophagosomes and autolysosomes, and sequestosome 1 (p62) degradation. Subsequent analysis indicated that RCE-4 activated the AMP-activated protein kinase (AMPK) pathway by upregulating AMPK and p-AMPK, and also inhibited the PI3K and extracellular signal-regulated kinase (ERK) signaling pathways by down-regulating p-PI3K, p-Akt, p-mTOR, Ras, c-Raf, p-c-Raf, dual specificity mitogen-activated protein kinase kinase (MEK)1/2, p-MEK1/2 and p-Erk1/2. Additionally, with increased treatment times RCE-4 may impair lysosomal cathepsin activity and inhibit autophagy flux by suppressing the expression of AMPK, p-AMPK, ULK1, p-ULK1 and p-Beclin-1, and upregulating that of p62. These results indicated that the dual RCE-4-induced inhibition of the PI3K and ERK pathways may result in a more significant anti-tumor 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 RCE-4. Taken together, RCE-4 induced autophagy to protect cancer cells against apoptosis, but AMPK-mediated autophagy was inhibited in the later stages of RCE-4 treatment. In addition, autophagy inhibition improved the therapeutic effect of RCE-4. These data highlight RCE-4 as a potential candidate for cervical cancer treatment.