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.

Identification of Fangjihuangqi Decoction as a late-stage autophagy inhibitor with an adjuvant anti-tumor effect against non-small cell lung cancer.

BACKGROUND: Clinically, although chemotherapy is one of the most commonly used methods of treating tumors, chemotherapeutic drugs can induce autophagic flux and increase tumor cell resistance, leading to drug tolerance. Therefore, theoretically, inhibiting autophagy may improve the efficacy of chemotherapy. The discovery of autophagy regulators and their potential application as adjuvant anti-cancer drugs is of substantial importance. In this study, we clarified that Fangjihuangqi Decoction (FJHQ, traditional Chinese medicine) is an autophagy inhibitor, which can synergistically enhance the effect of cisplatin and paclitaxel on non-small cell lung cancer (NSCLC) cells. METHODS: We observed the changes of autophagy level in NSCLC cells under the effect of FJHQ, and verified the level of the autophagy marker protein and cathepsin. Apoptosis was detected after the combination of FJHQ with cisplatin or paclitaxel, and NAC (ROS scavenger) was further used to verify the activation of ROS-MAPK pathway by FJHQ. RESULTS: We observed that FJHQ induced autophagosomes in NSCLC cells and increased the levels of P62 and LC3-II protein expression in a concentration- and time-gradient-dependent manner, indicating that autophagic flux was inhibited. Co-localization experiments further showed that while FJHQ did not inhibit autophagosome and lysosome fusion, it affected the maturation of cathepsin and thus inhibited the autophagic pathway. Finally, we found that the combination of FJHQ with cisplatin or paclitaxel increased the apoptosis rate of NSCLC cells, due to increased ROS accumulation and further activation of the ROS-MAPK pathway. This synergistic effect could be reversed by NAC. CONCLUSION: Collectively, these results demonstrate that FJHQ is a novel late-stage autophagy inhibitor that can amplify the anti-tumor effect of cisplatin and paclitaxel against NSCLC cells.

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.

Tanshinone IIA increases the bystander effect of herpes simplex virus thymidine kinase/ganciclovir gene therapy via enhanced gap junctional intercellular communication.

The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.