Identification of a novel autophagic inhibitor cepharanthine to enhance the anti-cancer property of dacomitinib in non-small cell lung cancer

OBJECTIVE Identification of novel autophagy inhibitors for the combinational treatment of non-small cell lung cancer(NSCLC).METHODS MTT assay and annexin V/PI staining assay were used to evaluate the cell proliferation and apoptosis,respectively.Immunofluorescence staining and cathepsin activity assay were used to detect autophagy.Small interfering RNA was performed to silence the genes and Western blot assay was used to evaluate the protein express levels.Xenograft experiments were applied for in vivo evaluation.RESULTS Cepharanthine,a natural compound,increased LC3-II expression and GFP-LC3 puncta formation in NSCLC NCI-H1975 cells.Numerous yellow puncta were observed in cepharanthine-treated cells with m RFP-EGFP-LC3 transfection.Co-staining of GFP-LC3 with LysoT racker red or LAMP1 antibody suggested that cepharanthine inhibits autophagosomes-lysosomes fusion.Moreover,cepharanthine attenuated the lysosomal cathepsins maturation.We also confirmed that dacomitinib induced cytoprotective autophagy.Combined treatment with cepharanthine increased the anti-cancer effects of dacomitinib in vitro and in vivo.Besides,cepharanthine could not enhance the anti-cancer effect of dacomitinib in autophagy deficient cells.CONCLUSION Cepharanthine might be further developed as a promising autophagic inhibitor,and combined treatment cepharanthine with dacomitinib could pose as an effective strategy for NSCLC treatment.

Natural Products in Cancer Therapy: Past, Present and Future

Natural products,with remarkable chemical diversity,have been extensively investigated for their anticancer potential for more than a half-century.The collective efforts of the community have achieved the tremendous advancements,bringing natural products to clinical use and discovering new therapeutic opportunities,yet the challenges remain ahead.With remarkable changes in the landscape of cancer therapy and growing role of cutting-edge technologies,we may have come to a crossroads to revisit the strategies to understand nature products and to explore their therapeutic utility.This review summarizes the key advancements in nature product-centered cancer research and calls for the implementation of systematic approaches,new pharmacological models,and exploration of emerging directions to revitalize natural products search in cancer therapy.

NQO1 dependent non-canonical necroptosis mediated by ROS and RIP1/RIP3 in parallel in glioma cancer cells

OBJECTIVE Glioblastomas(GBM) are the most malignant brain tumors in humans and have a very poor prognosis.New therapeutics are urgently needed.Here,we reported 2-methoxy-6-acetyl-7-methyljuglone(MAM)-induced cell death in U87 and U251 glioma cancer cells.METHODS Cells were cultured and treated with MAM,the cell viability was determined by MTT assay and LDH assay.Intracellular reactive oxygen species(ROS) generation was observed by DCF fluorescence.The protein expression was determined by Western blotting.RESULTS MAM induced glioma cancer cell death without caspase activation.The cell death induced by MAM was attenuated by the pharmacological or genetic blockage of necroptosis signaling,including RIP1 inhibitor necrostatin-1 s(Nec-1 s) and siRNA-mediated gene silencing of RIP1 and RIP3,but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1 H-Indol-3-yl)-3-pentylamino-maleimide(IM54).MAM treated U87 and U251 glioma cancer cells induced RIP1/RIP3 complex formation,ROS level increased,ATP concentration decreased and loss of plasma membrane integrity,further confirmed this process was necroptosis.The essential role of ROS was confirmed by the protective effect of ROS scavenger NAC.Interestingly,MAM induced necroptosis both triggered by RIP1/RIP3 complex and ROS generation.Moreover,MAM induced necroptosis through cytosolic calcium(Ca2 +) accumulation and sustained c-Jun N-terminal kinase(JNK) activation.Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate cell death.Further,we found there exists a feedback loop between RIP1 and JNK activation.Finally,MAM induced necroptosis was inhibited by dicoumarol(a NQO1 inhibitor).Dicoumarol exposed glioma cancer cells were resistant to RIP1/RIP3 complex formation and ROS generation.MAM induced necroptosis was independent of MLKL.CONCLUSION MAM induced non-canonical necroptosis through the NQO1-dependent ROS and RIP1/RIP3 pathway.This study also provided new insights into the molecular regulation of necroptosis in human glioma cancer cells and a promising approach for GBM treatment.

2-Methoxystypandrone induces iNOS expression by H_2O_2-dependent JNK activation for multiple cancer cell death

OBJECTIVE To investigate the mechanism of anticancer effect of 2-methoxystypandrone(2-MS),a natural naphthoquinone isolated from Polygonum cuspidatum Sieb.et Zucc.METHODSThree types of cancer cells were investigated in the research(A549,MCF7,B16-F10).Flow cytometer was used to determine ROS/RNS generation.Western blotting was used to detect related protein expression.Apoptosis assay,GSH/GSSG(reduced glutathione/oxidized glutathione)assay were performed using commercial kit.SiRNA knockdown was used to silence cj-un N-terminal kinase(JNK)and iNOS.High-performance liquid chromatography(HPLC)was used to detect the direct reaction of 2-MS with GSH.RESULTS 2-MS induced cytotoxity towards a panel of cancer cells,with less effect on normal cells.2-MS induced necroptosis in A549 cell and apoptosis in B16-F10 and MCF7cells.2-MS increased phosphorylation of JNK in three types of cancer cells.Inhibition of JNK with SP600125 or silencing JNK attenuated 2-MS-induced cell death.JNK also activated iNOS expression and led to nitric oxide(NO)generation in three cancer cells.NO-induced nitrative stress was responsible for DNA damage and necroptosis in A549 cells.NO also inhibited NF-κB expression and induced intrinsic apoptosis in B16-F10 and MCF7cells.Both NO scavenger hemoglobin and silencing iNOS can partially reverse 2-MS-induced cell death.Furthermore,we found that all of these were attributed to induction of hydrogen peroxide(H2O2),which was caused by glutathione(GSH)depletion through interaction of 2-MS with GSH.The interaction was validated through cell-free HPLC analysis.Both the H2O2 scavenger catalase and exogenous GSH can significantly reverse the 2-MS-induced cell death.But catalase did not protect against the decrease in GSH level.In contrast,there showed no clear increase of both H2O2 and NO in non-carcinoma liver cell LO2.CONCLUSION Taken together,a medicinal plant-derived 1,4-napthoquinone,induced iNOS expression by H2O2-dependent JNK activation,caused nitrative stress,finally led to cancer cell death by necroptosis or apoptosis.

CD47 blockade improves the therapeutic effect of osimertinib in non-small cell lung cancer

The third-generation epidermal growth factor receptor (EGFR) inhibitor osimertinib (OSI) has been approved as the first-line treatment for EGFR-mutant non-small cell lung cancer (NSCLC). This study aims to explore a rational combination strategy for enhancing the OSI efficacy. In this study, OSI induced higher CD47 expression, an important anti-phagocytic immune checkpoint, via the NF-κB pathway in EGFR-mutant NSCLC HCC827 and NCI-H1975 cells. The combination treatment of OSI and the anti-CD47 antibody exhibited dramatically increasing phagocytosis in HCC827 and NCI-H1975 cells, which highly relied on the antibody-dependent cellular phagocytosis effect. Consistently, the enhanced phagocytosis index from combination treatment was reversed in CD47 knockout HCC827 cells. Meanwhile, combining the anti-CD47 antibody significantly augmented the anticancer effect of OSI in HCC827 xenograft mice model. Notably, OSI induced the surface exposure of “eat me” signal calreticulin and reduced the expression of immune-inhibitory receptor PD-L1 in cancer cells, which might contribute to the increased phagocytosis on cancer cells pretreated with OSI. In summary, these findings suggest the multidimensional regulation by OSI and encourage the further exploration of combining anti-CD47 antibody with OSI as a new strategy to enhance the anticancer efficacy in EGFR-mutant NSCLC with CD47 activation induced by OSI.

Building on the backbone of CD47-based therapy in cancer:Combination strategies,mechanisms,and future perspectives

Described as a“don't eat me”signal,CD47 becomes a vital immune checkpoint in cancer.Its interaction with signal regulatory protein alpha(SIRPa)prevents macrophage phagocytosis.In recent years,a growing body of evidences have unveiled that CD47-based combination therapy exhibits a superior anti-cancer effect.Latest clinical trials about CD47 have adopted the regimen of collaborating with other therapies or developing CD47-directed bispecific antibodies,indicating the combination strategy as a general trend of the future.In this review,clinical and preclinical cases about the current combination strategies targeting CD47 are collected,their underlying mechanisms of action are discussed,and ideas from future perspectives are shared.

c-MYC-mediated TRIB3/P62^(+) aggresomes accumulation triggers paraptosis upon the combination of everolimus and ginsenoside Rh2

The mammalian target of rapamycin(m TOR) pathway is abnormally activated in lung cancer.However, the anti-lung cancer effect of m TOR inhibitors as monotherapy is modest. Here, we identified that ginsenoside Rh2, an active component of Panax ginseng C. A. Mey., enhanced the anti-cancer effect of the m TOR inhibitor everolimus both in vitro and in vivo. Moreover, ginsenoside Rh2 alleviated the hepatic fat accumulation caused by everolimus in xenograft nude mice models. The combination of everolimus and ginsenoside Rh2(labeled Eve-Rh2) induced caspase-independent cell death and cytoplasmic vacuolation in lung cancer cells, indicating that Eve-Rh2 prevented tumor progression by triggering paraptosis. EveRh2 up-regulated the expression of c-MYC in cancer cells as well as tumor tissues. The increased cMYC mediated the accumulation of tribbles homolog 3(TRIB3)/P62+ aggresomes and consequently triggered paraptosis, bypassing the classical c-MYC/MAX pathway. Our study offers a potential effective and safe strategy for the treatment of lung cancer. Moreover, we have identified a new mechanism of TRIB3/P62+ aggresomes-triggered paraptosis and revealed a unique function of c-MYC.

Garcinone E Blocks Autophagy Through Lysosomal Functional Destruction in Ovarian Cancer Cells

Background:High proliferative rate of cancer cells requires autophagy to maintain nutrient supply and intracellular homeostasis.As a result,impairing autophagic flux could be a novel strategy of cancer therapy.Aims and Objectives:In this study,the mechanism of a xanthone derivative isolated from Garcinia mangostana,garcinone E(GE),was investigated.Materials and Methods:Fluorescence assay was used to observe the accumulation and location of autophagosome and lysosome.Flow cytometry with Lyso-tracker red,MDC,and AO staining were applied to evaluate the lysosome accumulation and cellular acidity.Western blot and RT-qPCR were performed to evaluate the protein and mRNA levels,respectively.Results:GE could cause enhancement of LC3 II and p62 and the accumulation of autophagosome and lysosome.Meanwhile,it limited the protein level of Rab7,increased lysosomal pH,and inhibited the maturation of lysosomal hydrolases such as Cathepsin L,therefore blockaded the fusion of autophagosome and lysosome.Moreover,GE acted as a TFEB modulator by downregulating its protein level,which might contribute to autophagy dysfunction in ovarian cancer cells.Conclusions:GE interfered autophagosome–lysosome fusion in cancer cells,which demonstrated its application as an autophagy regulator and a potential therapeutic agent.

Cytotoxic cassane diterpenoids from the seeds of Caesalpinia sappan

Phytochemical investigation on the seeds of Caesalpinia sappan led to the isolation of five new cassanetype diterpenoids, named 11-oxo-phanginin A（1）, caesalsappanins O–Q（2–4） and phanginin U（5）,together with five known compounds. The structures of the new compounds were elucidated by extensive analysis of mass spectrometric and 1D and 2D NMR spectroscopic data. All the new compounds showed moderate cytotoxic effects on human breast cancer MCF-7 and human colon cancer HCT116 cell lines.

Discovery of a novel EGFR ligand DPBA that degrades EGFR and suppresses EGFR-positive NSCLC growth

Epidermal growth factor receptor(EGFR)activation plays a pivotal role in EGFR-driven non-small cell lung cancer(NSCLC)and is considered as a key target of molecular targeted therapy.EGFR tyrosine kinase inhibitors(TKIs)have been canonically used in NSCLC treatment.However,prevalent innate and acquired resistances and EGFR kinase-independent pro-survival properties limit the clinical efficacy of EGFR TKIs.Therefore,the discovery of novel EGFR degraders is a promising approach towards improving therapeutic efficacy and overcoming drug resistance.Here,we identified a 23-hydroxybetulinic acid derivative,namely DPBA,as a novel EGFR small-molecule ligand.It exerted potent in vitro and in vivo anticancer activity in both EGFR wild type and mutant NSCLC by degrading EGFR.Mechanistic studies disclosed that DPBA binds to the EGFR extracellular domain at sites differing from those of EGF and EGFR.DPBA did not induce EGFR dimerization,phosphorylation,and ubiquitination,but it significantly promoted EGFR degradation and repressed downstream survival pathways.Further analyses showed that DPBA induced clathrin-independent EGFR endocytosis mediated by flotillin-dependent lipid rafts and unaffected by EGFR TKIs.Activation of the early and late endosome markers rab5 and rab7 but not the recycling endosome marker rab11 was involved in DPBA-induced EGFR lysosomal degradation.The present study offers a new EGFR ligand for EGFR pharmacological degradation and proposes it as a potential treatment for EGFR-positive NSCLC,particularly NSCLC with innate or acquired EGFR TKI resistance.DPBA can also serve as a chemical probe in the studies on EGFR trafficking and degradation.