Verteporfin-loaded supramolecular micelles for enhanced cisplatin-based chemotherapy via autophagy inhibition

Cisplatin (CDDP) is one of the most successful chemotherapeutic agents for cancer therapy. However, CDDP can activate pro-survival autophagy which inhibits therapeutic efficacy of CDDP. Herein, autophagy inhibitor verteporfin (VTPF)...

Toosendanin, A Novel Late-Stage Autophagy Inhibitor Sensitizes Triple-Negative Breast Cancer to Irinotecan cChemotherapy In Vitro and In Vivo

Background: Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that develops resistance to chemotherapy frequently. Autophagy has been regarded as a pro-survival response to chemotherapeutic drugs in TNBC, and suppression of autophagy can be a therapeutic strategy to overcome drug resistance. Methods: We verified the efficacy of TSN in blocking autophagy flux. The co-localization of autophagosomes and lysosomes was analyzed. Then, lysosome function was determined by pH value and the activity of hydrolytic proteases. For preclinical research, human triple-negative breast cancer MDA-MB-231 and MDA-MB-436 were used for evaluating the anti-proliferative effect in vitro. In vivo, the nude mice were intraperitoneal injection of irinotecan (10 mg/kg), TSN (0.5 mg/kg) or a combination. Autophagy activity and cell apoptosis were determined in tumor tissue. The degree of pathological injury of tissue was evaluated by liver index.Results: Here we reported the natural autophagy inhibitor toosendanin, a triterpenoid extracted from Melia toosenda Sieb. et Zucc, potently inhibited late-stage autophagy in TNBC cells. This effect was achieved via elevating lysosome pH rather than blocking the fusion of autophagosomes and lysosomes. We further investigated the effects of toosendanin on the in vitro and in vivo TNBC models, in combination with chemotherapeutic drug irinotecan (or its active metabolite SN-38), a topoisomerase I (TOP1) inhibitor showing therapeutic potential for TNBC. The data showed that toosendanin blocked SN-38/irinotecan-induced protective autophagy, and significantly induced apoptosis in TNBC cells and tumor xenograft models when compared to SN-38/irinotecan or TSN treatment alone group.

Combining an Autophagy Inhibitor, MPT0L145, with Abemaciclib Is a New Therapeutic Strategy in GBM Treatment

Glioblastoma multiforme (GBM) is the most malignant brain tumor in the world, only 25% of GBM patients were alive one year after diagnosis. Although Temozolamide combined with radiation therapy more effectively prolonged the survival rate than radiation alone, the overall survival rate is still dismal. Therefore, a new therapeutic strategy is urgently needed. CDK4/6 inhibitors are newly FDA-approved agents to treat HR-positive, HER2-negative advanced, and metastatic breast cancers, and preclinical results showed that CDK4/6 inhibitors significantly reduced cell proliferation and tumor growth. However, several studies have suggested that CDK4/6 inhibitor-induced non-genetic changes caused treatment failure, including autophagy activation. Therefore, this study aimed to combine an autophagy inhibitor, MPT0L145, with abemaciclib to improve therapeutic efficiency. The use of abemaciclib effectively inhibited cell proliferation via suppression of RB phosphorylation and induced autophagy activation in GBM cancer cells. MPT0L145 treatment alone not only blocked autophagy activation, but also induced generation of ROS and DNA damage in a concentration-dependent manner. Importantly, MPT0L145 had a comparable penetration ability to TMZ in our blood brain barrier permeability assay. Combined MPT0L145 with abemaciclib significantly reduced cell proliferation, suppressed RB phosphorylation, and increased ROS production. In conclusion, the data suggested that blocking autophagy by MPT0L145 synergistically sensitized GBM cancer cells to abemaciclib and represents a potential therapeutic strategy for treating GBM in the future.

Comparative transcriptomic and metabolic profiling provides insight into the mechanism by which the autophagy inhibitor 3-MA enhances salt stress sensitivity in wheat seedlings

Background Salt stress hinders plant growth and production around the world. Autophagy induced by salt stress helps plants improve their adaptability to salt stress. However, the underlying mechanism behind this adaptability remains unclear. To obtain deeper insight into this phenomenon, combined metabolomics and transcriptomics analyses were used to explore the coexpression of differentially expressed-metabolite (DEM) and gene (DEG) between control and salt-stressed wheat roots and leaves in the presence or absence of the added autophagy inhibitor 3-methyladenine (3-MA). Results The results indicated that 3-MA addition inhibited autophagy, increased ROS accumulation, damaged photosynthesis apparatus and impaired the tolerance of wheat seedlings to NaCl stress. A total of 14,759 DEGs and 554 DEMs in roots and leaves of wheat seedlings were induced by salt stress. DEGs were predominantly enriched in cellular amino acid catabolic process, response to external biotic stimulus, regulation of the response to salt stress, reactive oxygen species (ROS) biosynthetic process, regulation of response to osmotic stress, ect. The DEMs were mostly associated with amino acid metabolism, carbohydrate metabolism, phenylalanine metabolism, carbapenem biosynthesis, and pantothenate and CoA biosynthesis. Further analysis identified some critical genes (gene involved in the oxidative stress response, gene encoding transcription factor (TF) and gene involved in the synthesis of metabolite such as alanine, asparagine, aspartate, glutamate, glutamine, 4-aminobutyric acid, abscisic acid, jasmonic acid, ect.) that potentially participated in a complex regulatory network in the wheat response to NaCl stress. The expression of the upregulated DEGs and DEMs were higher, and the expression of the down-regulated DEGs and DEMs was lower in 3-MA-treated plants under NaCl treatment. Conclusion 3-MA enhanced the salt stress sensitivity of wheat seedlings by inhibiting the activity of the roots and leaves, inhibiting autophagy in the roots and leaves, increasing the content of both H2O2 and O2•—, damaged photosynthesis apparatus and changing the transcriptome and metabolome of salt-stressed wheat seedlings.

Hydroxychloroquine synergizes with the PI3K inhibitor BKM120 to exhibit antitumor efficacy independent of autophagy

Background The critical role of phosphoinositide 3-kinase (PI3K) activation in tumor cell biology has prompted massive efforts to develop PI3K inhibitors (PI3Kis) for cancer therapy. However, recent results from clinical trials have shown only a modest therapeutic efficacy of single-agent PI3Kis in solid tumors. Targeting autophagy has controversial context-dependent effects in cancer treatment. As a FDA-approved lysosomotropic agent, hydroxychloroquine (HCQ) has been well tested as an autophagy inhibitor in preclinical models. Here, we elucidated the novel mechanism of HCQ alone or in combination with PI3Ki BKM120 in the treatment of cancer. Methods The antitumor effects of HCQ and BKM120 on three different types of tumor cells were assessed by in vitro PrestoBlue assay, colony formation assay and in vivo zebrafish and nude mouse xenograft models. The involved molecular mechanisms were investigated by MDC staining, LC3 puncta formation assay, immunofluorescent assay, flow cytometric analysis of apoptosis and ROS, qRT-PCR, Western blot, comet assay, homologous recombination (HR) assay and immunohistochemical staining. Results HCQ significantly sensitized cancer cells to BKM120 in vitro and in vivo. Interestingly, the sensitization mediated by HCQ could not be phenocopied by treatment with other autophagy inhibitors (Spautin-1, 3-MA and bafilomycin A1) or knockdown of the essential autophagy genes Atg5/Atg7, suggesting that the sensitizing effect might be mediated independent of autophagy status. Mechanistically, HCQ induced ROS production and activated the transcription factor NRF2. In contrast, BKM120 prevented the elimination of ROS by inactivation of NRF2, leading to accumulation of DNA damage. In addition, HCQ activated ATM to enhance HR repair, a high-fidelity repair for DNA double-strand breaks (DSBs) in cells, while BKM120 inhibited HR repair by blocking the phosphorylation of ATM and the expression of BRCA1/2 and Rad51. Conclusions Our study revealed that HCQ and BKM120 synergistically increased DSBs in tumor cells and therefore augmented apoptosis, resulting in enhanced antitumor efficacy. Our findings provide a new insight into how HCQ exhibits antitumor efficacy and synergizes with PI3Ki BKM120, and warn that one should consider the “off target” effects of HCQ when used as autophagy inhibitor in the clinical treatment of cancer.

Silica/OCP Affects the Viability of Osteoblasts through ROS-Induced Autophagy

Objective. To explore the effects of silicone gel nanoparticles modified with octacalcium phosphate on the surface (silica/OCP) polymer drugs on the proliferation of osteoblasts and autophagy. Method. Silica/OCP was prepared in vitro, and the quality of the sample preparation was tested through characterization experiments. The osteoblast cell line (hFOB1.19) was treated with silica/OCP, autophagy inhibitor (3-methyladenine (3-MA)), and silica/OCP+3-MA, respectively. The proliferation of hFOB1.19 cells was detected through the methylthiazolyldiphenyl-tetrazolium bromide (MTT) kit. Flow cytometry was used to detect the cell apoptosis. The change in protein beclin1 and P62 expression in hFOB1.19 cells was observed in Western blot. An ROS detection kit was used to detect the content of reactive oxygen species in hFOB1.19 cells. Results. Silica/OCP was a sphere with a particle size of 50 nm to 130 nm and had an OCP phase in electron projection microscopy and X-ray diffraction techniques. The results indicated that OCP successfully modified silica and the material was successfully prepared. An MTT kit and flow cytometry test showed that the cell viability of the cells treated with silica/OCP increased significantly ( P < 0.05 ), and the intracellular apoptosis phenomenon was significantly decreased ( P < 0.05 ) compared to the control group. Moreover, the inhibition of cell viability and promotion of apoptosis caused by the autophagy inhibitor 3-MA can be rescued. Western blotting demonstrated that the protein level of beclin1 in osteoblasts reached the highest after six hours of treatment with silica/OCP, and the protein level of p62, the substrate protein of autophagy, reached the lowest. At the same time, treatment of cells with the autophagy inhibitor 3-MA and silica/OCP+3-MA found that the protein levels of beclin1 and p62 in the silica/OCP+3-MA group were adjusted back compared to the 3-MA group. After adding the autophagy inhibitor, the reactive oxygen content in the cell was significantly increased ( P < 0.05 ) in the silica/OCP group. In the presence of intracellular reactive oxygen inhibitors catalase and silica/OCP, the cell viability of osteoblasts was significantly lower than that of the silica/OCP group but significantly higher than that of the silica/OCP+3-MA group. The apoptosis level of the silica/OCP+catalase group was also significantly lower than that of the silica/OCP+3-MA group ( P < 0.05 ) but was significantly higher than that of the silica/OCP group ( P < 0.05 ). Conclusion. Silica/OCP nanoparticles can upregulate the level of autophagy in osteoblasts and promote the proliferation of osteoblasts.

Heat treatment-induced autophagy promotes invasion and metastasis via TGF-β2 mediated epithelial-mesenchymal transition in breast cancer cells

Background Insufficient thermal ablation can cause accelerated malignant behaviors and increased metastasis in hepatocellular carcinoma (HCC), and epithelial-mesenchymal transition (EMT) and autophagy are implicated in tumor metastasis. However, whether interactions between autophagy and TGF-β2 induce EMT in breast cancer (BC) after insufficient microwave ablation (MWA) remains unclear. Methods In this study, we treated BC cells with sublethal heat treatment for simulating insufficient MWA, and then the effect of heat treatment on the BC cell phenotypes were explored. CCK-8, colony formation, flow cytometry, transwell and wound healing assays were performed to evaluate the influence of sublethal heat treatment on the proliferation, apoptosis, invasion and migration of BC cells treated with/without autophagy inhibitors. Western blotting, real-time quantitative PCR, immunofluorescence and transmission electron microscopy were carried out to determine the changes of markers associated with autophagy and EMT after sublethal heat treatment. Xenograft models in mice were established by using sublethal heat treated BC cells to investigate the effect of autophagy inhibitor on BC tumor growth in vivo. Results Results showed that heat treatment promoted the proliferation of survived BC cells, which was accompanied by autophagy induction. Heat treatment-induced autophagy up-regulated TGF-β2/Smad2 signaling and promoted phenotype of EMT, thereby enhancing abilities of migration and invasion in BC cells. Increase or decrease of TGF-β2 expression resulted in potentiation and suppression of autophagy as well as enhancement and abatement of EMT. Autophagy inhibitor facilitated apoptosis and repressed proliferation of BC cells in vitro, and thwarted BC cell tumor growth and pulmonary metastasis in vivo. Conclusions This study indicate that heat treatment-induced autophagy promotes invasion and metastasis via TGF-β2/Smad2-mediated EMT. Suppressing autophagy might be a new strategy for overcoming sufficient MWA caused progression and metastasis of residual BC cells.

Autophagic Schwann Cells Promote Perineural Invasion Mediated by the NGF/ATG7 Paracrine Pathway in Pancreatic Cancer

BackgroundPerineural invasion (PNI) and autophagy are two common features in the tumor microenvironment of pancreatic cancer (PanCa) and have a negative effect on prognosis. Potential mediator cells and the molecular mechanism underlying their relationships need to be fully elucidated. MethodsTo investigate the autophagy of Schwann cells (SCs) in PNI, we reproduced the microenvironment of PNI by collecting clinical PNI tissue, performing sciatic nerve injection of nude mice with cancer cells and establishing a Dorsal root ganglion（DRG） coculture system with cancer cell lines. Autophagy was detected by IHC, IF, transmission electron microscopy (TEM) and western blotting assays. Apoptosis was detected by IF, TEM and western blotting. NGF targeting molecular RO 08-2750（RO） and the autophagy inhibitor Chloroquine（CQ）were utilized to evaluate the effect on autophagy and apoptosis in SCs and PanCa cells in PNI samples.ResultsSC autophagy is activated in PNI by paracrine NGF from PanCa cells. Autophagy-activated Schwann cells promote PNI through a) enhanced migration and axon guidance toward PanCa cells and b) increased chemoattraction to PanCa cells. The NGF-targeting reagent RO and autophagy inhibitor CQ inhibited Schwann cell autophagic flux and induced Schwann cell apoptosis. Moreover, RO and CQ could induce PanCa cell apoptosis and showed good therapeutic effects in the PNI model.ConclusionsPanCa cells can induce autophagy in SCs through paracrine pathways such as the NGF/ATG7 pathway. Autophagic SCs exert a "nerve-repair like effect", induce a high level of autophagy of cancer cells, provide a "beacon" for the invasion of cancer cells to nerve fibers, and induce directional growth of cancer cells. Targeting NGF and autophagy for PNI treatment can block nerve infiltration and is expected to provide new directions and an experimental basis for the research and treatment of nerve infiltration in pancreatic cancer.