Critical role of heme oxygenase-1 in chaetoglobosin A by triggering reactive oxygen species mediated mitochondrial apoptosis in colorectal cancer.

The incidence rate of colorectal cancer (CRC) has been increasing and poses severe threats to human health worldwide and developing effective treatment strategies remains an urgent task. In this study, Chaetoglobosin A (ChA), an endophytic fungal metabolite from the medicinal herb-derived fungus Chaetomium globosum Km1126, was identified as a potent and selective antitumor agent in human CRC. ChA induced growth inhibition of CRC cells in a concentration-dependent manner but did not impair the viability of normal colon cells. ChA triggered mitochondrial intrinsic and caspase-dependent apoptotic cell death. In addition, apoptosis antibody array analysis revealed that expression of Heme oxygenase-1 (HO-1) was significantly increased by ChA. Inhibition of HO-1 increased the sensitivity of CRC cells to ChA, suggesting HO-1 may play a protective role in ChA-mediated cell death. ChA induced cell apoptosis via the induction of reactive oxygen species (ROS) and ROS scavenger (NAC) prevented ChA-induced cell death, mitochondrial dysfunction, and HO-1 activation. ChA promoted the activation of c-Jun N-terminal kinase (JNK), and co-administration of JNK inhibitor or siRNA markedly reversed ChA-mediated apoptosis. ChA significantly decreased the tumor growth without eliciting any organ toxicity or affecting the body weight of the CRC xenograft mice. This is the first study to demonstrate that ChA exhibits promising anti-cancer properties against human CRC both in vitro and in vivo. ChA is a potential therapeutic agent worthy of further development in clinical trials for cancer treatment.

Activation of multiple proteolysis systems contributes to acute cadmium cytotoxicity.

Cadmium exhibits both toxic and carcinogenic effects, and its cytotoxicity is linked to various cellular pathways, such as oxidative stress, ubiquitin-proteasome, and p53-mediated response pathways. The molecular mechanism(s) underlying cadmium cytotoxicity appears to be complex, but remains largely unclear. Here, we examined the effects of cadmium on the protein catabolism using two surrogate markers, DNA topoisomerases I and II alpha and its contribution to cytotoxicity. We have found that cadmium exposure induced time- and concentration-dependent decreases in the protein level of surrogate markers and therefore suggest that cadmium may be involved in proteolysis system activation. A pharmacological study further revealed the novel role(s) of these proteolytic activities and reactive oxygen species (ROS) in the cadmium-induced acute toxicity: (i) Proteasome inhibition only partially relieved the cadmium-induced proteolysis of topoisomerases; (ii) Moreover, we report for the first time that the activation of metalloproteases, serine proteases, and cysteine proteases contributes to the acute cadmium cytotoxicity; (iii) Consistent with the notion that both ROS generation and proteolysis system activation contribute to the cadmium-induced proteolysis and cytotoxicity, the scavenger N-acetylcysteine and aforementioned protease inhibition not only reduced the cadmium-induced topoisomerase degradation but also alleviated the cadmium-induced cell killing. Taken together, acute cadmium exposure may activate multiple proteolytic systems and ROS formation, subsequently leading to intracellular damage and cytotoxicity. Thus, our results provide a novel insight into potential action mechanism(s) by which cadmium exerts its cytotoxic effect and suggest potential strategies to prevent cadmium-associated acute toxicity.

Retraction notice to "Design, synthesis, and biological evaluation of heterotetracyclic quinolinone derivatives as anticancer agents targeting topoisomerases" [Eur. J. Med. Chem. 190 (2020) 112074].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The authors regret to inform that they would like to withdraw this accepted article, due to serious errors in authorship, affiliations, material sources and supporting grant names/numbers. The authors sincerely apologize for these oversights and miscommunications the study caused.

Design, synthesis, and biological evaluation of heterotetracyclic quinolinone derivatives as anticancer agents targeting topoisomerases.

A series of thiochromeno[2,3-c]quinolin-12-one derivatives with various substitutions were synthesized and evaluated as topoisomerase (Topo) inhibitors. Six (8, 10, 12, 14, 19, and 26) of 23 compounds showed strong inhibitory activities against Topo-mediated DNA relaxation and proliferation of five human cell lines including breast (MDA-MB-231, MDA-MB-468 and MCF7), colorectal (HCT116) and non-small cell lung (H1299) cancers. Among these, compounds 14 and 26 exhibited full inhibitory activities against Topo I at 3 µM and Topo IIα at 1 µM. Cancer cells treated with 26 accumulated DNA damage and were arrested at the G2/M phase. With time, cells proceeded to apoptosis, as revealed by increased amounts of cells with fragmented DNA and cleavage of caspase-8 and -9. In contrast, normal breast epithelial cells showed low sensitivity to 26. Taken together, our study identifies 26 as a potent Topo dual-inhibitor with low toxicity to normal cells, and elucidates that the terminal amino group of N-2-aminoethylamino or N-3-aminopropylamino at the 6th position and 8,10-di-halogen substituents on thiochromeno[2,3-c]quinolin-12-one are critical for the Topo-inhibiting and cancer-killing activities.

Inflammatory interferon activates HIF-1α-mediated epithelial-to-mesenchymal transition via PI3K/AKT/mTOR pathway.

BACKGROUND: Tumor microenvironments (TMEs) activate various axes/pathways, predominantly inflammatory and hypoxic responses, impact tumorigenesis, metastasis and therapeutic resistance significantly. Although molecular pathways of individual TME are extensively studied, evidence showing interaction and crosstalk between hypoxia and inflammation remain unclear. Thus, we examined whether interferon (IFN) could modulate both inflammatory and hypoxic responses under normoxia and its relation with cancer development. METHODS: IFN was used to induce inflammation response and HIF-1α expression in various cancer cell lines. Corresponding signaling pathways were then analyzed by a combination of pharmacological inhibitors, immunoblotting, GST-Raf pull-down assays, dominant-negative and short-hairpin RNA-mediated knockdown approaches. Specifically, roles of functional HIF-1α in the IFN-induced epithelial-mesenchymal transition (EMT) and other tumorigenic propensities were examined by knockdown, pharmacological inhibition, luciferase reporter, clonogenic, anchorage-independent growth, wound-healing, vasculogenic mimicry, invasion and sphere-formation assays as well as cellular morphology observation. RESULTS: We showed for the first time that IFN induced functional HIF-1α expression in a time- and dose- dependent manner in various cancer cell lines under both hypoxic and normoxic conditions, and then leading to an activated HIF-1α pathway in an IFN-mediated pro-inflammatory TME. IFN regulates anti-apoptosis activity, cellular metastasis, EMT and vasculogenic mimicry by a novel mechanism through mainly the activation of PI3K/AKT/mTOR axis. Subsequently, pharmacological and genetic modulations of HIF-1α, JAK, PI3K/AKT/mTOR or p38 pathways efficiently abrogate above IFN-induced tumorigenic propensities. Moreover, HIF-1α is required for the IFN-induced invasiveness, tumorigenesis and vasculogenic mimicry. Further supports for the HIF-1α-dependent tumorigenesis were obtained from results of xenograft mouse model and sphere-formation assay. CONCLUSIONS: Our mechanistic study showed an induction of HIF-1α and EMT ability in an IFN-mediated inflammatory TME and thus demonstrating a novel interaction between inflammatory and hypoxic TMEs. Moreover, targeting HIF-1α may be a potential target for inhibiting tumor tumorigenesis and EMT by decreasing cancer cells wound healing and anchorage-independent colony growth. Our results also lead to rationale guidance for developing new therapeutic strategies to prevent relapse via targeting TME-providing IFN signaling and HIF-1α programming.

Trichodermin induces c-Jun N-terminal kinase-dependent apoptosis caused by mitotic arrest and DNA damage in human p53-mutated pancreatic cancer cells and xenografts.

Pancreatic cancer is an aggressive malignancy, which generally responds poorly to chemotherapy. In this study, trichodermin, an endophytic fungal metabolite from Nalanthamala psidii, was identified as a potent and selective antitumor agent in human pancreatic cancer. Trichodermin exhibited antiproliferative effects against pancreatic cancer cells, especially p53-mutated cells (MIA PaCa-2 and BxPC-3) rather than normal pancreatic epithelial cells. We found that trichodermin induced caspase-dependent and mitochondrial intrinsic apoptosis. Trichodermin also increased apoptosis through mitotic arrest by activating Cdc2/cyclin B1 complex activity. Moreover, trichodermin promoted the activation of c-Jun N-terminal kinase (JNK), and inhibition of JNK by its inhibitor, shRNA, or siRNA significantly reversed trichodermin-mediated caspase-dependent apoptosis. Trichodermin triggered DNA damage stress to activate p53 function for executing apoptosis in p53-mutated cells. Importantly, we demonstrated that trichodermin with efficacy similar to gemcitabine, profoundly suppressed tumor growth through inducing intratumoral DNA damage and JNK activation in orthotopic pancreatic cancer model. Based on these findings, trichodermin is a potential therapeutic agent worthy of further development into a clinical trial candidate for treating cancer, especially the mutant p53 pancreatic cancer.

Rhapontigenin inhibits TGF-ß-mediated epithelial­mesenchymal transition via the PI3K/AKT/mTOR pathway and is not associated with HIF-1α degradation.

The epithelial-mesenchymal transition (EMT) is a pivotal event in cancer cell invasion and metastasis. Emerging evidence suggests that rhapontigenin (Rha) may impede the progression of cancer by disrupting angiogenesis and the EMT. However, the underlying mechanism of Rha has not yet been clarified. In this study, we used transforming growth factor ß (TGF-ß) to trigger EMT in diverse types of cancer cells and revealed that Rha inhibited TGF-ß-induced EMT and derived­cell invasiveness. The effects of TGF-ß were blocked by Rha via interference with the PI3K/AKT/mTOR/GSK3ß/ß­catenin signaling pathway. Furthermore, Rha also inhibited TGF-ß­induced expression of transcription regulators Snail and hypoxia-inducible factor 1α (HIF-1α) by causing their degradation by the 26S proteasome. Surprisingly, although HIF-1α was degraded with Snail as a result of Rha exposure, HIF-1α was not a key factor involved in TGF-ß-mediated EMT induced by Rha. Knocking-down Snail expression, but not HIF-1α expression, by RNA interference dramatically reversed TGF-ß-mediated EMT. Moreover, Rha abolished TGF-ß-triggered cell invasiveness. Our results demonstrate that Rha inhibits TGF-ß-induced EMT in cancer cells by suppressing the activity of the PI3K/AKT/mTOR pathway. Therefore, Rha may represent a new route for therapeutic intervention in cancer patients and merits future studies to assess its potential.

DNA topoisomerase III alpha regulates p53-mediated tumor suppression.

PURPOSE: Human DNA topoisomerase III alpha (hTOP3α) is involved in DNA repair surveillance and cell-cycle checkpoints possibly through formatting complex with tumor suppressors. However, its role in cancer development remained unsolved. EXPERIMENTAL DESIGN: Coimmunoprecipitation, sucrose gradient, chromatin immunoprecipitation (ChIP), real time PCR, and immunoblotting analyses were performed to determine interactions of hTOP3α with p53. Paired cell lines with different hTOP3α levels were generated via ectopic expression and short hairpin RNA (shRNA)-mediated knockdown approaches. Cellular tumorigenic properties were analyzed using cell counting, colony formation, senescence, soft agar assays, and mouse xenograft models. RESULTS: The hTOP3α isozyme binds to p53 and cofractionizes with p53 in gradients differing from fractions containing hTOP3α and BLM. Knockdown of hTOP3α expression (sh-hTOP3α) caused a higher anchorage-independent growth of nontumorigenic RHEK-1 cells. Similarly, sh-hTOP3α and ectopic expression of hTOP3α in cancer cell lines caused increased and reduced tumorigenic abilities, respectively. Genetic and mutation experiments revealed that functional hTOP3α, p53, and p21 are required for this tumor-suppressive activity. Mechanism-wise, ChIP data revealed that hTOP3α binds to the p53 and p21 promoters and positively regulates their expression. Two proteins affect promoter recruitments of each other and collaborate in p21 expression. Moreover, sh-hTOP3α and sh-p53 in AGS cells caused a similar reduction in senescence and hTOP3α mRNA levels were lower in gastric and renal tumor samples. CONCLUSION: We concluded that hTOP3α interacts with p53, regulates p53 and p21 expression, and contributes to the p53-mediated tumor suppression.

A negative feedback of the HIF-1α pathway via interferon-stimulated gene 15 and ISGylation.

PURPOSE: The IFN-stimulated gene 15 (ISG15)- and ubiquitin-conjugation pathways play roles in mediating hypoxic and inflammatory responses. To identify interaction(s) between these two tumor microenvironments, we investigated the effect of ISG15 on the activity of the master hypoxic transcription factor HIF-1α. EXPERIMENTAL DESIGN: IFN and desferoxamine treatments were used to induce the expression of ISGs and HIF-1α, respectively. Interactions between HIF-1α and the ISG15 and ISGylation system were studied using knockdown of mRNA expression, immunoblotting, coimmunoprecipitation, and pull-down analyses. Effects of the ISG15 and ISGylation system on the HIF-1α-directed processes were examined using reporter, reverse transcription polymerase chain reaction (RT-PCR), and tumorigenic growth assays. RESULTS: We found that the level of the free form of HIF-1α is differentially regulated by IFN treatment, and that the free ISG15 level is lower under hypoxia. Mechanism-directed studies have shown that HIF-1α not only interacts physically with ISG15, but is also ISGylated in multiple domains. ISG15 expression disrupts the functional dimerization of HIF-1α and -1ß. Subsequently, expression of the ISG15 and/or ISGylation system attenuates HIF-1α-mediated gene expression and tumorigenic growth. CONCLUSION: In summary, our results revealed cross-talk between inflammatory and hypoxic pathways through the ISGylation of HIF-1α. On the basis of these results, we propose a novel negative feedback loop for the HIF-1α-mediated pathway involving the regulation of HIF-1α via IFN-induced ISGylation.

TTK/hMps1 participates in the regulation of DNA damage checkpoint response by phosphorylating CHK2 on threonine 68.

CHK2/hCds1 plays important roles in the DNA damage-induced cell cycle checkpoint by phosphorylating several important targets, such as Cdc25 and p53. To obtain a better understanding of the CHK2 signaling pathway, we have carried out a yeast two-hybrid screen to search for potential CHK2-interacting proteins. Here, we report the identification of the mitotic checkpoint kinase, TTK/hMps1, as a novel CHK2-interacting protein. TTK/hMps1 directly phosphorylates CHK2 on Thr-68 in vitro. Expression of a TTK kinase-dead mutant, TTK(D647A), interferes with the G(2)/M arrest induced by either ionizing radiation or UV light. Interestingly, induction of CHK2 Thr-68 phosphorylation and of several downstream events, such as cyclin B1 accumulation and Cdc2 Tyr-15 phosphorylation, is also affected. Furthermore, ablation of TTK expression using small interfering RNA results not only in reduced CHK2 Thr-68 phosphorylation, but also in impaired growth arrest. Our results are consistent with a model in which TTK functions upstream from CHK2 in response to DNA damage and suggest possible cross-talk between the spindle assembly checkpoint and the DNA damage checkpoint.