Marsdenia tenacissima extract induces endoplasmic reticulum stress-associated immunogenic cell death in non-small cell lung cancer cells through targeting AXL

Ethnopharmacological relevance: Marsdenia Tenacissima (Roxb.) Wight et Arn. is a traditional Chinese medicine. Its standardized extract (MTE), with the trade name Xiao-Ai-Ping injection, is widely used for Cancer treatment. The pharmacological effects of MTE-inducing Cancer cell death have been primarily explored. However, whether MTE triggers tumor endoplasmic reticulum stress (ERS)-associated immunogenic cell death (ICD) is unknown.

Aim of the study: To determine the potential role of endoplasmic reticulum stress in the anti-cancer effects of MTE, and uncover the possible mechanisms of endoplasmic reticulum stress-associated immunogenic cell death induced by MTE.

Material and methods: The anti-tumor effects of MTE on non-small cell lung Cancer (NSCLC) were examined through CCK-8 and wound healing assay. Network pharmacology analysis and RNA-sequencing (RNA seq) were performed to confirm the biological changes of NSCLCs after MTE treatment. Western blot, qRT-PCR, Reactive Oxygen Species (ROS) assay, and mitochondrial membrane potential (MMP) assay were used to explore the occurrence of endoplasmic reticulum stress. Immunogenic cell death-related markers were tested by ELISA and ATP release assay. Salubrinal was used to inhibit the endoplasmic reticulum stress response. SiRNA and bemcentinib (R428) were used to impede the function of Axl. Axl phosphorylation was regained by recombinant human Gas6 protein (rhGas6). The effects of MTE on endoplasmic reticulum stress and immunogenic cell death response were also proved in vivo. The Axl inhibiting compound in MTE was explored by molecular docking and confirmed by Western blot.

Results: MTE inhibited cell viability and migration of PC-9 and H1975 cells. Enrichment analysis identified that differential genes after MTE treatment were significantly enriched in endoplasmic reticulum stress-related biological processes. MTE decreased mitochondrial membrane potential (MMP) and increased ROS production. Meanwhile, endoplasmic reticulum stress-related proteins (ATF6, GRP-78, ATF4, XBP1s, and CHOP) and immunogenic cell death-related markers (ATP, HMGB1) were upregulated, and the Axl phosphorylation level was suppressed after MTE treatment. However, when salubrinal (an endoplasmic reticulum stress inhibitor) and MTE were co-treated cells, the inhibitory effects of MTE on PC-9 and H1975 cells were impaired. Importantly, inhibition of Axl expression or activity also promotes the expression of endoplasmic reticulum stress and immunogenic cell death-related markers. Mechanistically, MTE induced endoplasmic reticulum stress and immunogenic cell death by suppressing Axl activity, and these effects were attenuated when Axl activity recovered. Moreover, MTE significantly increased the expression of endoplasmic reticulum stress-related markers in LLC tumor-bearing mouse tumor tissues and plasma levels of ATP and HMGB1. Molecular docking illustrated that kaempferol has the strongest binding energy with Axl and suppresses Axl phosphorylation.

Conclusion: MTE induces endoplasmic reticulum stress-associated immunogenic cell death in NSCLC cells. The anti-tumor effects of MTE are dependent upon endoplasmic reticulum stress. MTE triggers endoplasmic reticulum stress-associated immunogenic cell death by inhibiting Axl activity. Kaempferol is an active component that inhibits Axl activity in MTE. The present research revealed the role of Axl in regulating endoplasmic reticulum stress and enriched the anti-tumor mechanisms of MTE. Moreover, kaempferol may be considered a novel Axl Inhibitor.

AXL; Endoplasmic reticulum; Immunogenic cell death; Kaempferol; Marsdenia tenacissima extract; Non-small cell lung cancer.