Three chromomycin derivatives, chromomycins A3 (1, CA3), A5 (2, CA5), and monodeacetylchromomycin A3 (3, MDA-CA3), were identified from the soil-derived Streptomyces sp. CGMCC 26516. A reinvestigation of the structure of CA5 is reported, of which the absolute configuration was unambiguously determined for the first time to be identical with that of CA3 based on nuclear magnetic resonance (NMR) data analysis as well as NMR and electronic circular dichroism calculations. Compounds 1-3 showed potent cytotoxicity against the non-small-cell lung cancer (NSCLC) cells (A549, H460, H157-c-FLIP, and H157-LacZ) and down-regulated the protein expression of c-FLIP in A549 cells. The IC50 values of chromomycins in H157-c-FLIP were higher than that in H157-LacZ. Furthermore, si-c-FLIP promoted anti-proliferation effect of chromomycins in NSCLC cells. In nude mice xenograft model, 1 and 2 both showed more potent inhibition on the growth of H157-lacZ xenografts than that of H157-c-FLIP xenografts. These results verify that c-FLIP mediates the anticancer effects of chromomycins in NSCLC.

Triple-negative breast cancer (TNBC) is an aggressive subtype with poor prognosis of breast cancer. Thiostrepton exerts anti-tumor activities against several cancers including TNBC. Herein we discussed the new molecular mechanisms of thiostrepton in TNBC. Thiostrepton inhibited MDA-MB-231 cell viability, accompanied by a decrease of c-FLIP and p-SMAD2/3. c-FLIP overexpression reduced the sensitivity of MDA-MB-231 cells to thiostrepton, while SMAD2/3 knockdown increased the sensitivity of MDA-MB-231 cells to thiostrepton. Moreover, c-FLIP overexpression significantly increased the expression and phosphorylation of SMAD2/3 proteins and vice versa. In conclusion, our study reveals c-FLIP/SMAD2/3 signaling pathway as a novel mechanism of antitumor activity of thiostrepton.

Metastatic lung adenocarcinoma (LuAC) presents a significant clinical challenge due to the short latency and the lack of efficient treatment options. Therefore, identification of molecular vulnerabilities in metastatic LuAC holds great importance in the development of therapeutic drugs against this disease. In this study, we performed a genome-wide siRNA screening using poorly and highly brain-metastatic LuAC cell lines. Using this approach, we discovered that compared to poorly metastatic LuAC (LuAC-Par) cells, brain-metastatic LuAC (LuAC-BrM) cells exhibited a significantly higher vulnerability to c-FLIP (an inhibitor of caspase-8)-depletion-induced apoptosis. Furthermore, in vivo studies demonstrated that c-FLIP knockdown specifically inhibited growth of LuAC-BrM, but not the LuAC-Par, tumors, suggesting the addiction of LuAC-BrM to the function of c-FLIP for their survival. Our in vitro and in vivo analyses also demonstrated that LuAC-BrM is more sensitive to c-FLIP-depletion due to ER stress-induced activation of the c-JUN and subsequent induction of stress genes including ATF4 and DDIT3. Finally, we found that c-JUN not only sensitized LuAC-BrM to c-FLIP-depletion-induced cell death but also promoted brain metastasis in vivo, providing strong evidence for c-JUN\'s function as a double-edged sword in LuAC-BrM. Collectively, our findings not only reveal a novel link between c-JUN, brain metastasis, and c-FLIP addiction in LuAC-BrM but also present an opportunity for potential therapeutic intervention.

BACKGROUND: Defects in apoptosis regulation are one of the classical features of cancer cells, often associated with more aggressiveness and failure to therapeutic options. We investigated the combinatorial antitumor effects of a natural product, physachenolide C (PCC) and bortezomib, in KRASmut/P53mut lung cancer cells and xenograft mice models.
METHODS: The in vitro anticancer effects of the bortezomib and PCC combination were investigated using cell viability, migration, and invasion assays in 344SQ, H23, and H358 cell lines. Furthermore, the effects of combination treatment on the critical parameters of cellular metabolism, including extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation based on the oxygen consumption rate of cancer cells were assessed using Seahorse assay. Finally, the antitumor effect of the bortezomib (1 mg/kg) and PCC (10 mg/kg) combination was evaluated using xenograft mice models.
RESULTS: Our data showed that the bortezomib-PCC combination was more effective in reducing the viability of lung cancer cells in comparison with the individual treatments. Similarly, the combination treatment showed a significant inhibition of cell migration and invasion of cancer cells. Additionally, the key anti-apoptotic protein c-FLIP was significantly inhibited along with a substantial reduction in the key parameters of cellular metabolism in cancer cells. Notably, the bortezomib or PCC inhibited the tumor growth compared to the control group, the tumor growth inhibition was much more effective when bortezomib was combined with PCC in tumor xenograft mice models.
CONCLUSIONS: These findings demonstrate that PCC sensitizes cancer cells to bortezomib, potentially improving the antitumor effects against KRASmut/P53mut lung cancer cells, with an enhanced efficacy of combination treatments without causing significant side effects.

The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-Associated Death Domain) following death receptors\' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents a promising anticancer agent, as it selectively induces apoptosis in transformed cells without altering the cellular machinery of healthy cells. Unfortunately, the presence of TRAIL resistance mechanisms in a variety of cancer types represents a major hurdle, thus limiting the use of TRAIL as a single agent. Accumulating studies have shown that TRAIL-mediated apoptosis can be facilitated in resistant tumors by combined treatment with antitumor agents, ranging from synthetic molecules to natural products. Among the latter, flavonoids, the most prevalent polyphenols in plants, have shown remarkable competence in improving TRAIL-driven apoptosis in resistant cell lines as well as tumor-bearing mice with minimal side effects. Here, we summarize the molecular mechanisms, such as the upregulation of death receptor (DR)4 and DR5 and downregulation of key anti-apoptotic proteins [e.g., cellular FLICE-inhibitory protein (c-FLIP), X-linked inhibitor of apoptosis protein (XIAP), survivin], underlying the TRAIL-sensitizing properties of different classes of flavonoids (e.g., flavones, flavonols, isoflavones, chalcones, prenylflavonoids). Finally, we discuss limitations, mainly related to bioavailability issues, and future perspectives regarding the clinical use of flavonoids as adjuvant agents in TRAIL-based therapies.

BACKGROUND: Patients with metastatic triple-negative breast cancer (mTNBC) have a higher probability of developing visceral metastasis within 5 years after the initial diagnosis. Therefore, a deeper understanding of the progression and spread of mTNBC is urgently needed.
METHODS: The isobaric tag for relative and absolute quantitation (iTRAQ)-based LC-MS/MS proteomic approach was applied to identify novel membrane-associated proteins in the lung-tropic metastatic cells. Public domain datasets were used to assess the clinical relevance of the candidate proteins. Cell-based and mouse models were used for biochemical and functional characterization of the protein molecule Sciellin (SCEL) identified by iTRAQ to elucidate its role and underlying mechanism in promoting lung colonization of TNBC cells.
RESULTS: The iTRAQ-based LC-MS/MS proteomic approach identified a membrane-associated protein SCEL that was overexpressed in the lung-tropic metastatic cells, and its high expression was significantly correlated with the late-stage TNBC and the shorter survival of the patients. Downregulation of SCEL expression significantly impaired the 3D colony-forming ability but not the migration and invasion ability of the lung colonization (LC) cells. Knockdown of SCEL reduced TNF-α-induced activation of the NF-κB/c-FLIP pro-survival and Akt/Erk1/2 growth signaling pathways in the LC cells. Specifically, knockdown of SCEL expression switched TNF-α-mediated cell survival to the caspase 3-dependent apoptosis. Conversely, ectopic expression of SCEL promoted TNF-α-induced activation of NF-κB/c-FLIP pro-survival and Akt/Erk1/2 pro-growth signaling pathway. The result of co-immunoprecipitation (Co-IP) and GST pull-down assay showed that SCEL could interact with TNFR1 to promote its protein stability. The xenograft mouse model experiments revealed that knockdown of SCEL resulted in increase of caspase-3 activity, and decrease of ki67 and TNFR1 expression as well as increase of tumor-associated macrophages in the metastatic lung lesions. Clinically, SCEL expression was found to be positively correlated with TNFR1 in TNBC tissues. Lastly, we showed that blocking TNF-α-mediated cell survival signaling by adalimumab effectively suppressed the lung colonization of the SCEL-positive, but not the SCEL-downregulated LC cells in the tail-vein injection model.
CONCLUSIONS: Our findings indicate that SCEL plays an essential role in the metastatic lung colonization of TNBC by promoting the TNF-α/TNFR1/NF-κB/c-FLIP survival and Akt/Erk1/2 proliferation signaling. Thus, SCEL may serve as a biomarker for adalimumab treatment of TNBC patients.

Tubeimoside-1 (TBMS-1), a traditional Chinese medicinal herb, is commonly used as an anti-cancer agent. In this study, we aimed to investigate its effect on the sensitization of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Our results revealed that even though monotherapy using TBMS-1 or TRAIL at sublethal concentrations did not affect cancer cell death, combination therapy using TBMS-1 and TRAIL increased apoptotic cell death. Mechanistically, TBMS-1 destabilized c-FLIP expression by downregulating STAMBPL1, a deubiquitinase (DUB). Specifically, when STAMBPL1 and c-FLIP bound together, STAMBPL1 deubiquitylated c-FLIP. Moreover, STAMBPL1 knockdown markedly increased sensitivity to TRAIL by destabilizing c-FLIP. These findings were further confirmed in vivo using a xenograft model based on the observation that combined treatment with TBMS-1 and TRAIL decreased tumor volume and downregulated STAMBPL1 and c-FLIP expression levels. Overall, our study revealed that STAMBPL1 is essential for c-FLIP stabilization, and that STAMBPL1 depletion enhances TRAIL-mediated apoptosis via c-FLIP downregulation.

Excessive apoptosis of intestinal epithelial cell (IEC) is a crucial cause of disrupted epithelium homeostasis, leading to the pathogenesis of ulcerative colitis (UC). The regulation of Takeda G protein-coupled receptor-5 (TGR5) in IEC apoptosis and the underlying molecular mechanisms remained unclear, and the direct evidence from selective TGR5 agonists for the treatment of UC is also lacking. Here, we synthesized a potent and selective TGR5 agonist OM8 with high distribution in intestinal tract and investigated its effect on IEC apoptosis and UC treatment. We showed that OM8 potently activated hTGR5 and mTGR5 with EC50 values of 202 ± 55 nM and 74 ± 17 nM, respectively. After oral administration, a large amount of OM8 was maintained in intestinal tract with very low absorption into the blood. In DSS-induced colitis mice, oral administration of OM8 alleviated colitis symptoms, pathological changes and impaired tight junction proteins expression. In addition to enhancing intestinal stem cell (ISC) proliferation and differentiation, OM8 administration significantly reduced the rate of apoptotic cells in colonic epithelium in colitis mice. The direct inhibition by OM8 on IEC apoptosis was further demonstrated in HT-29 and Caco-2 cells in vitro. In HT-29 cells, we demonstrated that silencing TGR5, inhibition of adenylate cyclase or protein kinase A (PKA) all blocked the suppression of JNK phosphorylation induced by OM8, thus abolished its antagonizing effect against TNF-α induced apoptosis, suggesting that the inhibition by OM8 on IEC apoptosis was mediated via activation of TGR5 and cAMP/PKA signaling pathway. Further studies showed that OM8 upregulated cellular FLICE-inhibitory protein (c-FLIP) expression in a TGR5-dependent manner in HT-29 cells. Knockdown of c-FLIP blocked the inhibition by OM8 on TNF-α induced JNK phosphorylation and apoptosis, suggesting that c-FLIP was indispensable for the suppression of OM8 on IEC apoptosis induced by OM8. In conclusion, our study demonstrated a new mechanism of TGR5 agonist on inhibiting IEC apoptosis via cAMP/PKA/c-FLIP/JNK signaling pathway in vitro, and highlighted the value of TGR5 agonist as a novel therapeutic strategy for the treatment of UC.

OBJECTIVE: Further investigation on the mechanism of action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in NSCLC would shed light on the understanding of TRAIL resistance and provide new clues for the counter-strategy.
BACKGROUND: Cellular FLICE-inhibitory protein (c-FLIP) is a critical inhibitor of TRAIL-induced apoptosis. Our previous study suggested that glycogen synthase kinase 3β (GSK3β) positively regulated c-FLIP expression in human lung adenocarcinoma cells. Meanwhile, other studies reported that c-FLIP was degraded by HECT-type E3 ligase ITCH (Itchy E3 Ubiquitin Protein Ligase) via the proteasome pathway.
OBJECTIVE: We will explore whether ITCH is involved in the expression regulation of c-FLIP positively controlled by GSK3β during the treatment of TRAIL.
METHODS: Human lung adenocarcinoma cells were used to stably overexpress and knockdown GSK3β. Quantitative real-time PCR (qRT-PCR) assay was used to test the expressional level of mRNA of genes. Western blot analysis was employed to detect the expression of proteins at the protein level. siRNA of ITCH was used to knock down its expression. TRAIL treatment was used to cause apoptosis.
RESULTS: In the present study, we have confirmed the degradation of c-FLIP by ITCH protein and the downregulation of ITCH expression by GSK3β in lung adenocarcinoma cells. Moreover, ITCH silencing reversed the downregulation of c-FLIP protein caused by GSK3β-knockdown in the cells. Accordingly, TRAIL-induced apoptosis facilitated by GSK3β knockdown was blocked by the combined interference of ITCH.
CONCLUSIONS: These results suggested that GSK3β/ITCH axis regulated the stability of c-FLIP and influenced TRAIL-induced apoptosis. Taken together, our study revealed a GSK3β/ITCH/c-FLIP axis, which counteracts TRAIL-induced apoptosis in human lung adenocarcinoma cells.