Triptolide inhibits epithelial ovarian tumor growth by blocking the hedgehog/Gli pathway.

Epithelial ovarian cancer (EOC), the most predominant subtype of ovarian cancer (OC), involves poor prognosis and exhibits high aggression. Triptolide (TPL), like other Chinese herbs, has historically played a significant role in modern medicine. The screening system based on Gli-dependent luciferase reporter activity assessed the effects of over 800 natural medicinal materials on hedgehog (Hh) signaling pathway activity and discovered that TPL had an excellent inhibitory effect on Hh signaling pathway activity. However, the significance and mechanism of TPL involvement in regulating the Hh pathway have not been well explored. Thus, this work aimed to understand better how TPL affects the Hh pathway activity, which, in turn, influences the biological behavior of EOC. Our findings observed that Smo agonist SAG-induced EOC cell proliferation, migration, and invasion were drastically reversed by TPL in a concentration-dependent pattern. Further evidence suggested that TPL promotes the degradation of Gli1 and Gli2 to inhibit the activity of the Hh signaling pathway by relying on Gli1 and Gli2 ubiquitination. Our in vivo studies also confirmed that TPL could significantly inhibit the tumor growth of EOC. Taken together, our results revealed that one of the antitumor mechanisms of TPL was the targeted inhibition of the Hh/Gli pathway.

A comprehensive analysis of PANoptosome to prognosis and immunotherapy response in pan-cancer.

PANoptosis, a programmed cell death, shares key characteristics of apoptosis, pyroptosis, and necroptosis. Accumulating evidence suggests that PANoptosis plays a crucial role in tumorigenesis. However, the respective regulation mechanisms in cancer are so far unclear. Using various bioinformatic approaches, we comprehensively analyzed the expression patterns, genetic alterations, prognostic value, and immunological role of PANoptosis genes in pan-cancer. Expression of the PANoptosis gene, PYCARD, was validated based on the Human Protein Atlas database and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). We found that PANoptosis genes were aberrantly expressed in most cancer types, which was consistent with the validation of PYCARD expression. Concurrently, PANoptosis genes and PANoptosis scores were significantly associated with patient survival in 21 and 14 cancer types, respectively. Pathway analysis showed that PANoptosis score was positively correlated with pathways linked to immune and inflammatory responses in pan-cancer, such as IL6-JAK-STAT3 signaling, the interferon-gamma response, and IL2-STAT5 signaling. In addition, the PANoptosis score was significantly correlated with the tumor microenvironment, the infiltration levels of most immune cells (i.e.NK cells, CD8+ T cells, CD4+ T cells, DC cells), and immune-related genes. Furthermore, it was a predictive indicator of immunotherapy response in patients with tumors. These insights substantially improve our understanding of PANoptosis components in cancers and may inspire the discovery of novel prognostic and immunotherapy response biomarkers.

Hedgehog-Gli2 Signaling Promotes Chemoresistance in Ovarian Cancer Cells by Regulating MDR1.

BACKGROUND: Cisplatin (DDP) resistance remains a key challenge in improving the clinical outcome of patients with ovarian cancer (OC). Gli2 overexpression can lead to DDP resistance in OC cells, but the specific underlying regulatory mechanism remains unclear. The membrane transporter encoding gene MDR1 positively regulates chemotherapy resistance in various cancer types. We evaluated MDR1 as a potential Gli2 downstream target and the contribution of the Gli2/MDR1 axis in promoting DDP resistance in OC cells. METHODS: To generate drug-resistant SKOV3/DDP cells, SKOV3 cells were grown for six months under continuous induction wherein the DDP concentration was steadily increased. Gli2 expression in OC cells with varying DDP sensitivities was detected using western blot. Cell counting kit-8 assays were used to assess the DDP sensitivity of SKOV3, SKOV3/DDP, A2780, and A2780/DDP cells and reversal of DDP resistance in SKOV3/DDP and A2780/DDP cells. Cell proliferation was analyzed using 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. The transcriptional regulation of MDR1 by Gli2 was determined using luciferase reporter assays. Finally, xenograft OC tumors were generated in nude mice, which were then treated with intraperitoneal DDP or phosphate-buffered saline (PBS) injections to investigate if Gli2 affected DDP resistance in OC in vivo. RESULTS: DDP-resistant SKOV3/DDP and A2780/DDP cells showed higher expression of Gli2 and MDR1 as compared with that in DDP-sensitive OC cells. Gli2 knockdown in SKOV3/DDP cells significantly reduced MDR1 expression, whereas it increased DNA damage, thereby sensitizing OC cells to DDP. Similar results were obtained after targeting Gli2 expression with the Gli-antagonist 61 inhibitor (GANT61) in SKOV3/DDP and A2780/DDP cells. In cells stably overexpressing Gli2, treatment with gradient concentrations of verapamil, an MDR1 inhibitor, significantly inhibited MDR1 expression. Our findings indicate that downregulation of MDR1 expression may reverse OC cell resistance to DDP. Moreover, dual-luciferase reporter gene assays confirmed that MDR1 is a direct downstream target of Gli2, with Gli2 positively regulating MDR1 expression. Finally, subcutaneous xenotransplantation in nude mice demonstrated that Gli2 plays a key role in regulating OC drug resistance. CONCLUSIONS: We identified a mechanism by which Hedgehog-Gli signaling regulates OC chemoresistance by modulating MDR1 expression. Hence, Gli2 and MDR1 are potential biomarkers and therapeutic targets in patients with chemoresistant OC.

The Hedgehog signaling pathway promotes chemotherapy resistance via multidrug resistance protein 1 in ovarian cancer.

Various studies have revealed that the Hedgehog (Hh) signaling pathway promotes ovarian cancer invasion, migration and drug resistance. Previous studies by our group have identified a set of genes, including multidrug resistance gene 1 (MDR1), that are regulated by Hh signaling in ovarian cancer. However, the association between Hh signaling activation and MDR1 expression requires further validation. In the present study, reverse transcription­quantitative PCR or western blot assays were used to evaluate the mRNA and protein expression levels of MDR1, Sonic Hh (Shh), glioma­associated oncogene 2 (Gli2), Gli1 and γ­phosphorylated H2A.X variant histone (γ­H2AX). MTT and colony­formation assays were performed to determine the effect of cisplatin (DDP) after inhibiting the Hh pathway in ovarian cancer cells. The results indicated that MDR1, Gli2 and Shh levels were much higher in SK­OV­3 cells with acquired DDP resistance than in native SK­OV­3 cells. ES­2 cells with overexpression of Gli2 were capable of efficiently forming colonies in the presence of low DDP concentrations. By contrast, Gli2 knockdown in SK­OV­3 cells decreased the colony­forming ability under the same concentration of DDP. As determined by MTT assays, knockdown of Gli2 or targeting of the Hh signaling pathway with either Gli­antagonist 61 (GANT61) or cyclopamine, in combination with DDP treatment, diminished the viability of ES­2 and SK­OV­3 cells, whereas Gli2 overexpression increased the viability of ES­2 cells in the presence of DDP. Knockdown of Gli2 or targeting the Hh signaling pathway with GANT61 also increased γ­H2AX levels but decreased the expression of MDR1 in the presence of DDP. MDR1 expression is regulated by the Hh signaling pathway and is likely a downstream transcription factor of Gli2. In conclusion, targeting the Hh signaling pathway increases the sensitivity of ovarian cancer to DDP. MDR1 is a target gene of the Hh signaling pathway and this pathway may affect chemoresistance of ovarian cancer to DDP via MDR1.