Identification of Fangchinoline as a novel autophagy inhibitor with an adjuvant of chemotherapy against lung cancer.

Autophagy is a fundamental recycling pathway that enhances cellular resilience, promoting survival. However, this survival mechanism can impede anti-cancer treatment strategies designed to induce cell death. In this study, we identified a novel autophagy inhibitor, Fangchinoline (Fan) isolated from the traditional Chinese medicine Stephania tetrandra. We speculated that when Fan blocks autophagy, cancer cells lose substantial self-preservation abilities during treatment. Firstly, we examined in detail the mechanism through which Fan inhibits autophagy. Specifically, Fan induced a significant increase in autophagosomes, as indicated by GFP-LC3 labeling, confirmed by the up-regulation of LC3-II. The autophagy receptor protein p62 was also up-regulated, suggesting a potential inhibition of autophagy flux. We further ruled out the possibility of fusion barriers between lysosomes and autophagosomes, as confirmed by their co-localization in double fluorescence staining. However, the lysosomal acid environment might be compromised, as suggested by the diminished fluorescence of acidity-sensitive dyes in the lysosomes and the corresponding decrease in mature forms of lysosomal cathepsin. To test the anti-cancer potential of Fan, we combined it with Cisplatin (Cis) or Paclitaxel (PTX) for lung cancer cell treatment. This combined treatment demonstrated a synergistically enhanced killing effect. These promising anti-tumor results were also replicated in a xenografted tumor model. The significance of this research lies in the identification of Fan as a potent autophagy inhibitor and its potential to enhance the efficacy of existing anti-cancer drugs. By unraveling the mechanisms of Fan's action on autophagy and demonstrating its synergistic effect in combination therapies, our study provides valuable insights for developing novel strategies to overcome autophagy-mediated resistance in cancer treatment.

Berbamine Hydrochloride inhibits lysosomal acidification by activating Nox2 to potentiate chemotherapy-induced apoptosis via the ROS-MAPK pathway in human lung carcinoma cells.

Autophagy is typically activated in cancer cells as a rescue strategy in response to cellular stress (e.g., chemotherapy). Herein, we found that Berbamine Hydrochloride (Ber) can act as an effective inhibitor of the late stage of autophagic flux, thereby potentiating the killing effect of chemotherapy agents. Lung carcinoma cells exposed to Ber exhibited increased autophagosomes, marked by LC3-II upregulation. The increased level of p62 after Ber treatment indicated that the autophagic flux was blocked at the late stage. The lysosome staining assay and cathepsin maturation detection indicated impaired lysosomal acidification. We found that Nox2 exhibited intensified co-localization with lysosomes in Ber-treated cells. Nox2 is a key enzyme for superoxide anion production capable of transferring electrons into the lysosomal lumen, thereby neutralizing the inner protons; this might explain the aberrant acidification. This hypothesis is further supported by the observed reversal of lysosomal cathepsin maturation by Nox2 inhibitors. Finally, Ber combined with cisplatin exhibited a synergistic killing effect on lung carcinoma cells. Further data suggested that lung carcinoma cells co-treated with Ber and cisplatin accumulated excessive reactive oxygen species (ROS), which typically activated MAPK-mediated mitochondria-dependent apoptosis. The enhanced anti-cancer effect of Ber combined with cisplatin was also confirmed in an in vivo xenograft mouse model. These findings indicate that Ber might be a promising adjuvant for enhancing the cancer cell killing effect of chemotherapy via the inhibition of autophagy. In this process, Nox2 might be a significant mediator of Ber-induced aberrant lysosomal acidification.

Tubeimoside I-induced lung cancer cell death and the underlying crosstalk between lysosomes and mitochondria.

Cancer cells have developed chemoresistance and have improved their survival through the upregulation of autophagic mechanisms that protect mitochondrial function. Here, we report that the traditional Chinese anticancer agent tubeimoside I (Tub), which is a potent inhibitor of autophagy, can promote mitochondria-associated apoptosis in lung cancer cells. We found that Tub disrupted both mitochondrial and lysosomal pathways. One of its mechanisms was the induction of DRP1-mediated mitochondrial fragmentation. Another mechanism was the blocking of late-stage autophagic flux via impairment of lysosomal acidification through V-ATPase inhibition; this blocks the removal of dysfunctional mitochondria and results in reactive oxygen species (ROS) accumulation. Excessive ROS accumulation causes damage to lysosomal membranes and increases lysosomal membrane permeability, which leads to the leakage of cathepsin B. Finally, cathepsin B upregulates Bax-mediated mitochondrial outer membrane permeability and, subsequently, cytosolic cytochrome C-mediated caspase-dependent apoptosis. Thus, the cancer cell killing effect of Tub is enhanced through the formation of a positive feedback loop. The killing effect of Tub on lung cancer cells was verified in xenografted mice. In summary, Tub exerts a dual anticancer effect that involves the disruption of mitochondrial and lysosomal pathways and their interaction and, thereby, has a specific and enhanced killing effect on lung cancer cells.

Hederagenin potentiated cisplatin- and paclitaxel-mediated cytotoxicity by impairing autophagy in lung cancer cells.

Autophagy inhibition has been demonstrated to increase the efficacy of conventional chemotherapy. In this study, we identified hederagenin, a triterpenoid derived from Hedera helix, as a potent inhibitor of autophagy and then hypothesized that hederagenin might synergize with chemotherapeutic drugs (e.g., cisplatin and paclitaxel) to kill lung cancer cells. Firstly, we observed that hederagenin induced the increased autophagosomes in lung cancer cells concomitantly with the upregulation of LC3-II and p62, which indicated the impairment of autophagic flux. The colocalization assay indicated hederagenin could not block the fusion of lysosomes and autophagosomes, whereas the lysosomal acidification might be inhibited by hederagenin as revealed by the reduced staining of acidity-sensitive reagents (i.e., Lysotracker and acridine orange). The aberrant acidic environment then impaired the function of lysosome, which was evidenced by the decrease of mature cathepsin B and cathepsin D. Lastly, hederagenin, in agree with our hypothesis, promoted pro-apoptotic effect of cisplatin and paclitaxel with the accumulation of reactive oxygen species (ROS); while the synergistic effect could be abolished by the ROS scavenger, N-acetyl-L-cysteine. These data summarily demonstrated hederagenin-induced accumulation of ROS by blocking autophagic flux potentiated the cytotoxicity of cisplatin and paclitaxel in lung cancer cells.

Screening and Identification of Potential Biomarkers for Hepatocellular Carcinoma: An Analysis of TCGA Database and Clinical Validation.

INTRODUCTION: Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. Up to now, many genes associated with HCC have not yet been identified. In this study, we screened the HCC-related genes through the integrated analysis of the TCGA database, of which the potential biomarkers were also further validated by clinical specimens. The discovery of potential biomarkers for HCC provides more opportunities for diagnostic indicators or gene-targeted therapies. METHODS: Cancer-related genes in The Cancer Genome Atlas (TCGA) HCC database were screened by a random forest (RF) classifier based on the RF algorithm. Proteins encoded by the candidate genes and other associated proteins obtained via protein-protein interaction (PPI) analysis were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The newly identified genes were further validated in the HCC cell lines and clinical tissue specimens by Western blotting, immunofluorescence, and immunohistochemistry (IHC). Survival analysis verified the clinical value of genes. RESULTS: Ten genes with the best feature importance in the RF classifier were screened as candidate genes. By comprehensive analysis of PPI, GO and KEGG, these genes were confirmed to be closely related to HCC tumors. Representative NOX4 and FLVCR1 were selected for further validation by biochemical analysis which showed upregulation in both cancer cell lines and clinical tumor tissues. High expression of NOX4 or FLVCR1 in cancer cells predicts low survival. CONCLUSION: Herein, we report that NOX4 and FLVCR1 are promising biomarkers for HCC that may be used as diagnostic indicators or therapeutic targets.

Dioscin Inhibits the Invasion and Migration of Hepatocellular Carcinoma HepG2 Cells by Reversing TGF-ß1-Induced Epithelial-Mesenchymal Transition.

Dioscin is a natural steroidal saponin that can be isolated from Chinese medicine, such as Dioscoreae rhizoma. It has wild range of pharmacological activities such as hepatoprotection, a lipid-lowering effect, and anti-inflammation. Recently, mounting studies reported the anticancer effect of dioscin on a variety of tumor cells. However, the potential effect of dioscin on the epithelial-mesenchymal transition (EMT) of HepG2 cells is unclear. In the present study, dioscin was identified to inhibit transforming growth factor-ß1 (TGF-ß1) and induced invasive and migratory behavior of HepG2 cells. Consistently, the expression of the epithelial marker E-cadherin and gap junction proteins increased following dioscin treatment, while mesenchymal markers decreased, including N-cadherin, Vimentin, Snail, and Slug. Furthermore, we discovered that TGF-ß1 induces phosphorylation of JNK, p38, and Erk, whereas the activation of these kinases was reversed by dioscin treatment in a dose-dependent manner. With the addition of Asiatic acid, a p38 activator, the inhibitory effect of dioscin on EMT was reversed. Taken together, these data indicated that dioscin inhibits EMT in HepG2 cells, which is mediated in large part by inhibition of the p38-MAPK signaling.

Antitumor effects of circ-EPHB4 in hepatocellular carcinoma via inhibition of HIF-1α.

The protein EPHB4 plays a vital role in various tumor types. However, few studies into the function of circ-EPHB4 (hsa_circ_0001730) in tumors have been conducted. This study aimed to investigate the functions of circ-EPHB4 and the underlying mechanism of circ-EPHB4 in regulating hepatocellular carcinoma (HCC). The expression of circ-EPHB4 was found to be downregulated in HCC tumor tissues, whereas circ-EPHB4 overexpression suppressed cell viability, induced apoptosis, and inhibited cell migration and invasion in Huh7 and HepG2 cells. circ-EPHB4 levels were negatively correlated with tumor weight, size, and metastasis foci in nude mouse models, suggesting circ-EPHB4 inhibits tumorigenesis, tumor development, and metastasis. In addition, HIF-1α and PI3K-AKT pathways were markedly affected by circ-EPHB4 overexpression. HIF-1α could potentially be the target of circ-EPHB4. By overexpressing both HIF-1α and circ-EPHB4, the antitumor effect of circ-EPHB4 should be most probably correlated with HIF-1α. In conclusion, circ-EPHB4 is a tumor inhibitor in HCC and functions by inhibiting HIF-1α expression.

Resveratrol enhances the radiosensitivity of nasopharyngeal carcinoma cells by downregulating E2F1.

Identification of safe, effective radiosensitizing agents is urgently needed to improve the outcome of radiotherapy in nasopharyngeal cancer (NPC). In this study, we assessed the ability of the polyphenol resveratrol to act as a radiosensitizer in vitro and in vivo. CNE-1 cells were treated with 50 µM resveratrol for 24 h, then irradiated. E2F transcription factor 1 (E2F1) was stably knocked down and overexpressed using lentiviruses. A xenograft model of NPC was established in nude mice using CNE-1 cells. Compared to control DMSO­treated CNE-1 cells, resveratrol inhibited colony-forming ability and induced G1 phase cell cycle arrest. Radiation survival curves confirmed resveratrol significantly sensitized CNE-1 cells, and resveratrol in combination with 2 Gy irradiation synergistically increased apoptosis. Immunoblotting showed resveratrol dose- and time-dependently downregulated E2F1 and phospho-AKT (p-AKT). Knockdown of E2F1 significantly increased radiosensitivity and downregulated p-AKT; overexpression of E2F1 reversed resveratrol-induced radiosensitivity and upregulated p-AKT. In vivo, 50 mg/kg/day resveratrol and 4 Gy irradiation led to significantly lower tumor volume and tumor weight compared to resveratrol or irradiation alone. Our findings show that resveratrol increases the radiosensitivity of NPC cells by downregulating E2F1 and inhibiting p-AKT, and therefore has potential as a radiosensitizer for NPC.

Tanshinone IIA arrests cell cycle and induces apoptosis in 786-O human renal cell carcinoma cells.

Tanshinone IIA (Tan IIA), isolated from the Chinese medicinal herb Danshen, has been reported to have anticancer effects in several tumor models, while its effects on renal cell carcinoma have not been studied. In the present study, we evaluated the effects of Tan IIA on growth inhibition and apoptosis in the renal cancer cell line 786-O and its mechanism of action. Results of the MTT assay indicated that the treatment of 786-O cells with Tan IIA resulted in a concentration-dependent decrease in cell viability. Flow cytometry analysis revealed that Tan IIA treatment caused apoptosis following cell cycle perturbation. Furthermore, we examined the expression of cell cycle and apoptosis-related proteins using immunoblotting, which indicated an upregulation of p53, p21, bax and caspase-3 in Tan IIA-treated cells compared with Tan IIA-untreated cells. These results suggest that the activation of p53 and the upregulation of its target genes, including p21 and bax, may be involved in the mitochondrial apoptosis induced by Tan IIA in 786-O cells.