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 (1β, 3β, 5β, 25S)‑spirostan‑1, 3‑diol 1‑[α‑L‑rhamnopyranosyl‑(1→2)‑β‑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 downregulating 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.