In this study, the regulatory effects of CHA on the expression of programmed cell death ligand 1 (PD-L1) in esophageal squamous cell carcinoma (ESCC), as well as the role of interferon γ (IFN-γ), has been discussed using both in vitro and in vivo animal models. ESCC murine model was established according to the standard operating procedures (SOP) of Animal Experiment Center of Institute of Materia Medica, Chinese Academy of Medical Sciences. The expression of PD-L1 in esophageal tissues of murine models was analyzed using the microarray assay. Then, the results were verified by qRT-PCR, Western blot and immunohistochemistry (IHC) staining, the molecular mechanism was explored in KYSE180 and KYSE510 ESCC cells in vitro. The results showed that CHA could suppress the expression of PD-L1 in tumor tissues in murine models significantly, rather than the expression in KYSE180 and KYSE510 ESCC cells in vitro. However, after the pretreatment of IFN-γ, the expression of PD-L1 was significantly increased, then it was down-regulated by CHA in both doseand time-dependent manner. Meanwhile, the expression of interferon regulatory factor 1 (IRF1), an upstream regulatory factor of PD-L1, was suppressed by CHA in both KYSE180 and KYSE510 pretreated with IFN-γ, which was consistent with the expression of PD-L1. These results indicate that CHA down-regulates the expression of PD-L1 in ESCC via IFN-γ-IRF1 signaling pathway, providing the molecular theoretical basis for exploration of new treatment of ESCC.

As a potential cancer immunotherapeutic agent, CHA has entered phase II clinical trials in China as a lyophilized powder formulation for treating glioma. However, the in vivo instability of CHA necessitates daily intramuscular injections, resulting in patient noncompliance. In this study, CHA-phospholipid complex (PC)-containing PEGylated liposomes (CHA-PC PEG-Lipo, named as CPPL), with CHA-PC as the drug intermediate, were prepared to lower the administration frequency. CPPL demonstrated excellent physicochemical properties, enhanced tumor accumulation, and inhibited tumor growth even when the administration interval was prolonged to 4 days when compared to a CHA solution and CHA-PC loaded liposomes (CHA-PC Lipo, labeled as CPL), both of which only demonstrated antitumor efficacy with once-daily administration.

These findings indicate that CHA-encapsulated mannosylated liposomes have great potential to enhance the immunotherapeutic efficacy of CHA by inducing a shift from the M2 to the M1 phenotype.

This review summarizes the pharmacological effects as well as the underlying mechanisms of CHA, providing an important theoretical basis for further research on CHA drug targets and potential mechanisms.

In this study, we confirmed the inhibition of proliferation by CHA in ESCC cells, as well as the reduction of ESCC xenograft volume by CHA in vivo. In addition, CHA also suppressed both the migration and invasion of ESCC cells in vitro. In a carcinogen-induced murine model of ESCC, hyperplasia of the esophagus was slowed by CHA, while mice suffering from ESCC that were treated with CHA had longer survival times than mice in the control group. The measurement of pluripotency factors (BMI1, SOX2, OCT4 and Nanog) that are related to poor prognosis revealed reduced expression of both BMI1 and SOX2, but not of OCT4 or Nanog, in ESCC cells, in both a dose- and time-dependent manner.

CHA might be a safe and effective differentiation-inducer for cancer therapy. “Educating”cancer cells to differentiate, rather than killing them, could be a novel therapeutic strategy for cancer.