Andrographolide Promotes Ferroptosis in Pancreatic Cancer via Targeting and Activating HSP90/GPX4 Ubiquitination.

Timosaponin AIII (Tim-AIII), a steroid saponin, exhibits strong anticancer activity in a variety of cancers, especially breast cancer and liver cancer. However, the underlying mechanism of the effects of Tim-AIII-mediated anti-lung cancer effects remain obscure. In this study, we showed that Tim-AIII suppressed cell proliferation and migration, induced G2/M phase arrest and ultimately triggered cell death of non-small cell lung cancer (NSCLC) cell lines accompanied by the release of reactive oxygen species (ROS) and iron accumulation, malondialdehyde (MDA) production, and glutathione (GSH) depletion. Interestingly, we found that Tim-AIII-mediated cell death was reversed by ferroptosis inhibitor ferrostatin-1 (Fer-1). Meanwhile, the heat shock protein 90 (HSP90) was predicted and verified as the direct binding target of Tim-AIII by SwissTargetPrediction (STP) and surface plasmon resonance (SPR) assay. Further study showed that Tim-AIII promoted HSP90 expression and Tim-AIII induced cell death was blocked by the HSP90 inhibitor tanespimycin, indicating that HSP90 was the main target of Tim-AIII to further trigger intracellular events. Mechanical analysis revealed that the Tim-AIII-HSP90 complex further targeted and degraded glutathione peroxidase 4 (GPX4), and promoted the ubiquitination of GPX4, as shown by an immunoprecipitation, degradation and in vitro ubiquitination assay. In addition, Tim-AIII inhibited cell proliferation, induced cell death, led to ROS and iron accumulation, MDA production, GSH depletion, as well as GPX4 ubiquitination and degradation, were markedly abrogated when HSP90 was knockdown by HSP90-shRNA transfection. Importantly, Tim-AIII also showed a strong capacity of preventing tumor growth by promoting ferroptosis in a subcutaneous xenograft tumor model, whether C57BL/6J or BALB/c-nu/nu nude mice. Together, HSP90 was identified as a new target of Tim-AIII. Tim-AIII, by binding and forming a complex with HSP90, further targeted and degraded GPX4, ultimately induced ferroptosis in NSCLC. These findings provided solid evidence that Tim-AIII can serve as a potential candidate for NSCLC treatment.

Andrographolide induces ferroptosis in colorectal cancer via P53-mediated downregulation of the SLC7A11/GPX4 signaling pathway.

Peroxiredoxins (Prxs) are a ubiquitously expressed family of thiol peroxidases that reduce hydrogen peroxide, peroxynitrite, and hydroperoxides using a highly conserved cysteine. There is substantial evidence that oxidative stress elicited by amyloid beta (Abeta) accumulation is a causative factor in the pathogenesis of Alzheimer disease (AD). Here we show that Abeta-resistant PC12 cell lines exhibit increased expression of multiple Prx isoforms with reduced cysteine oxidation. Abeta-resistant PC12 cells also display higher levels of thioredoxin and thioredoxin reductase, two enzymes critical for maintaining Prx activity. PC12 cells and rat primary hippocampal neurons transfected with wild type Prx1 exhibit increased Abeta resistance, whereas mutant Prx1, lacking a catalytic cysteine, confers no protection. Using an antibody that specifically recognizes sulfinylated and sulfonylated Prxs, it is demonstrated that primary rat cortical nerve cells exposed to Abeta display a time-dependent increase in cysteine oxidation of the catalytic site of Prxs that can be blocked by the addition of the thiol-antioxidant N-acetylcysteine. In support of previous findings, expression of Prx1 is higher in post-mortem human AD cortex tissues than in age-matched controls. In addition, two-dimensional gel electrophoresis and mass spectrometry analysis revealed that Prx2 exists in a more oxidized state in AD brains than in control brains. These findings suggest that increased Prx expression and resistance to sulfhydryl oxidation in Abeta-resistant nerve cells is a compensatory response to the oxidative stress initiated by chronic pro-oxidant Abeta exposure.

The Hirshberg Foundation for Pancreatic Cancer Research was founded by Agi Hirshberg in 1997, following her husband Ronald Hirshberg’s 8-month, 7-day battle with the disease. (http://www.pancreatic.org/). At the time, the Hirshberg family viewed pancreatic cancer as the ‘‘unknown enemy’’ (http:// www.pancreatic.org/site/c.htJYJ8MPIwE/b.8276835/k.66CB/ Agi_Hirshbergs_Story.htm). In the years since Ronald Hirshberg’s death, Agi Hirshberg has made it her mission to unmask the unknown for others facing the same enemy. When patients present with a new diagnosis of pancreatic cancer, most are already in an advanced stage of the disease. Pancreatic cancer is resistant to most chemotherapeutic agents and radiotherapy. These factors combine to make pancreatic cancer a highly lethal disease, and according to the National Cancer Institute (NCI) 2013 estimates, pancreatic cancer was diagnosed in approximately 45,220 US citizens, and some 38,460 died of the disease. 1 The Annual Report to the Nation on the Status of Cancer, 1975Y2009, shows that overall cancer death rates continued to decline in the United States among both men and women, among all major racial and ethnic groups, and for all of the many common cancer sites, including lung, colon and rectum, female breast, and prostate. However, this trend does not hold true for pancreatic cancer. The report demonstrates that death rates continued to increase during the latest time period (2000 through 2009) for pancreatic cancer. 2 Only about 23% of patients with cancer of the pancreas will still be living 1 year after diagnosis, and only about 4% will survive 5 years. Even for those diagnosed with pancreatic cancer that has not spread to other organs or systems, the overall 5-year survival rate approaches only 15%. The Hirshberg Foundation’s vision is to advance pancreatic cancer research and provide information, resources, and support to pancreatic cancer patients and their families. The mission of the Foundation is as follows: & To find a cure for pancreatic cancer in honor of Ron Hirshberg and the thousands of people who are diagnosed with this disease each year. & To create a premier Pancreatic Cancer Center where all needs of pancreatic cancer patients can be met in one location with the most advanced treatment options. & To be recognized as a patient support reference source for

Heat shock protein 25 (HSP25) interferes negatively with apoptosis through several pathways that involve its direct interaction with cytochrome c or Akt. Here we show that HSP25 inhibits protein kinase C (PKC) delta-mediated cell death through direct interaction. HSP25 binds to kinase-active PKCdelta to inhibit its kinase activity and translocation to the membrane, which results in reduced cell death. Deletion constructs of HSP25 and PKCdelta identified amino acids 90-103 of HSP25 and the C-terminal V5 region of PKCdelta as binding sites. In addition, the interaction between HSP25 and PKCdelta induced HSP25 phosphorylation at Ser-15 and Ser-86, and these phosphorylations permitted HSP25 release from PKCdelta. Based on these observations, we propose that after PKCdelta activation, HSP25 binds to the exposed V5 region of PKCdelta. This novel function of HSP25 accounts for its cytoprotective properties via the inhibition of PKCdelta and the enhancement of HSP25 phosphorylation.

Members of the yeast p24 family, including Emp24p and Erv25p, exist as heteromeric complexes that have been proposed to cycle between the endoplasmic reticulum (ER) and Golgi compartments. The specific functions and sites of action of p24 proteins are still unknown. Here we identified a human homolog of the yeast p24 family of proteins, named ERS25 (endoplasmic reticulum stress-response protein 25), and investigated its role in stress response. ERS25 is predicted to have an ER localization signal peptide, a GOLD (Golgi dynamics) domain, which is found in several eukaryotic Golgi and lipid-trafficking proteins, a coiled-coil region, and a transmembrane domain. We demonstrate that ERS25 is localized to the ER and is induced by ER-specific stress, heat shock, and oxidative stress. The selective induction of ERS25 by brefeldin A, but not tunicamycin, implicates the involvement of ERS25 in protein trafficking between the ER and the Golgi. Small interfering RNA-mediated inhibition of ERS25 results in a significant decrease in apoptosis as well as a reduction of reactive oxygen species induced by oxidative stress. Moreover, ERS25 depletion results in a significant increase in the levels of the ER chaperone HSP70 in response to heat-shock stress through increased levels of HSF-1. We also found that inhibition of ERS25 induction in response to heat shock enhanced the binding of HSP70 to Apaf-1, which is likely to interfere in stress-mediated apoptosis. Together, the data presented here demonstrate that ERS25 may play a critical role in regulation of heat-shock response and apoptosis.

Reactive oxygen species in the normal and acutely injured liver

Heat shock proteins (HSPs) are chaperone proteins that protect living cells against injury-inducing stimuli. Dysregulated expression of HSPs has been observed in various disease conditions including cancer. Using knock-out and transgenic animal approach, as well as standard, methods of heat shock protein 70 (HSP70) induction (i.e. thermal stress and arsenite administration), it has been shown that HSP70 protects against cell injury and acinar necrosis in experimental model of pancreatitis in animals. Animals in which HSP70 is induced prior to cerulein administration in a cerulein model of pancreatitis have reduced severity of pancreatitis, as demonstrated by lower serum amylase, lesser acinar necrosis on histology and decreased neutrophilic infiltration, suggesting that HSP70 is protective against cell death. Similar to the protective role of HSP70 in a pancreatitis model, HSP70 overexpression has been observed in pancreatic cancer and is believed to protect cancer cells from cell death. HSP70 is overexpressed both at mRNA and protein levels in pancreatic cancer cell lines as compared to normal pancreatic ductal cells. On a more clinical note, HSP70 is present in great abundance in pancreatic cancer clinical specimens as compared to normal pancreatic margins. Inhibition of HSP70 expression in pancreatic cancer cells leads to caspase-dependent apoptotic cell death and is a novel therapeutic modality for pancreatic cancer. Triptolide is a pharmacological agent which highly effective inhibiting HSP70 expression in pancreatic cancer cells and thus induces cell death. Moreover, triptolide is highly efficacious in reducing growth as well as locoregional spread of pancreatic tumors in an orthotopic model of pancreatic cancer and has tremendous potential as a novel therapeutic agent.

Background & Aims: The new therapy for pancreatic cancer which has the poorest prognosis among malignant tumors is high demand nowadays. The overall 5-year survival rate in all stages of this type of cancer is less than 5%, even with the multimodality therapy, including surgery, chemotherapy and radiotherapy. Although gemcitabine (GEM) is a key drug in the treatment of advanced pancreatic cancer, the therapeutic effect is limited because of drug resistance. Heat shock protein 90 (HSP90) is known to be overexpressed in several types of cancer cells, and its inhibition has shown promise in the treatment of solid malignancies. Src kinases work as a signal switch for multiple molecular signal transduction pathways. The objective of this study is to evaluate an antitumor effect of three kinds of small molecular weight compounds (antimetabolite Gemcitabine, HSP90 inhibitor NVP-AUY922, and Src kinase inhibitor Dasatinib) on tumor growth and chemotaxis in pancreatic cancer cell lines. Methods: Pancreatic cancer cell lines were treated with each of the 3 compounds to examine the effect on proliferation assay, apoptosis assay, Western blotting, and chemotaxis assay. Results: Of these compounds, HSP90 inhibitor inhibited cell proliferation in pancreatic cancer cells best in a dose- and time-dependent manner. Growth inhibitory effect of HSP90 inhibitor for pancreatic cancer cells were highest of the three. Further, HSP90 inhibitor inhibited the growth of gemcitabine sensitive and resistant pancreatic cancer cell lines. With flowcytometry and immunostaining, HSP90 inhibitor had higher apoptosis-inducing effect than gemcitabine. In the chemotaxis assay, HSP90 inhibitor inhibited the migration of pancreatic cancer cell best in the three compounds. In western blotting, HSP90 inhibitor suppressed the expression of multiple receptor tyrosine kinase proteins as well as the signal transduction in PI3K-Akt and RAS-MAPK pathways. Conclusion: In this study, HSP90 inhibitor was the best of 3 small molecular compounds and has proven effective in gemcitabine sensitive and resistant pancreatic cancer. The HSP90 inhibitor is useful as an antitumor drug by inhibiting proliferation and chemotaxis of pancreatic cancer cells through multiple kinase pathways. Citation Format: Masahiro Yamamura, Akira Yamauchi, Naoki Katase, Masakiyo Sakaguchi, Yosuke Katata, Hiroaki Tanioka, Makoto Okawaki, Takeshi Nagasaka, Yoshiyuki Yamaguchi. Comparing effects of small molecular weight compounds on proliferation and chemotaxis of pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2188.

We previously showed that activation of the A1 adenosine receptor protected the kidney against ischemia-reperfusion injury by induction and phosphorylation of heat shock protein 27 (HSP27). Here, we used mice that overexpress human HSP27 (huHSP27) to determine if kidneys from these mice were protected against injury. Proximal tubule cells cultured from the transgenic mice had increased resistance to peroxide-induced necrosis compared to cells from wild-type mice. However, after renal ischemic injury, HSP27 transgenic mice had decreased renal function compared to wild-type mice, along with increased renal expression of mRNAs of pro-inflammatory cytokines (TNF-alpha, ICAM-1, MCP-1) and increased plasma and kidney keratinocyte-derived cytokine. Following ischemic injury, neutrophils infiltrated the kidneys earlier in the transgenic mice. Flow cytometric analysis of lymphocyte subsets showed that those isolated from the kidneys of transgenic mice had increased CD3(+), CD4(+), CD8(+), and NK1.1(+) cells 3 h after injury. When splenocytes or NK1.1(+) cells were isolated from transgenic mice and adoptively transferred into wild-type mice there was increased renal injury. Further, depletion of lymphocytes by splenectomy or neutralization of NK1.1(+) cells resulted in improved renal function in the transgenic mice following reperfusion. Our study shows that induction of HSP27 in renal tubular cells protects against necrosis in vitro, but its systemic increase counteracts this protection by exacerbating renal and systemic inflammation in vivo.

Purpose: Pancreatic cancer (PC) is a deadly disease. Chemotherapy options are limited to gemcitabine and 5-fluorouracil (5-FU). The molecular profile of PC offers several growth receptors (epidermal growth factor receptor [EGFR], insulin-like growth factor 1 receptor [IGF-1R]) and tyrosine kinases (AKT and MAPK) as promising therapy targets. EGFR, IGF-1R, AKT and MAPK are dependent upon the heat shock protein 90 (HSP90) to gain the mature functional active protein form hence HSP90 represents a promising therapy target in PC. Functional inhibition of HSP90 has the advantage of disruption of many oncogenic signalling pathways simultaneously. The aim of this study was evaluate the efficacy of different HSP90 inhibitors in gemcitabine and 5-FU resistant PC. Material and Methods: Human gemcitabine and 5-FU resistant pancreatic cancer cell lines 5061, 5072 and 5156 were generated and brought in to culture from patients who were operated for PC at our institution and received gemcitabine as adjuvant therapy. The commercial L3.6pl cell line served as a control. Antiproliferative efficacy of three different HSP90 inhibitors (17AAG, 17DMAG and 17AEPGA) was evaluated by the MTT assay. Alterations in signalling pathway effectors and apoptosis upon HSP90 inhibition was determined by western blot analysis (WB). Results: The cell lines 5061, 5072 and 5156 were resistant to gemcitabine and 5-FU. In contrast, 17AAG and the water-soluble derivates 17DMAG and 17AEPGA displayed high antiproliferative activity in all tested cell lines. The calculated GI50 was below 1 µM. In line with the proliferation inhibition in the MTT assay, the expression of EGFR, IGF-1R, AKT and MAPK was significantly down regulated under HSP90 inhibition as verified inWB. In contrast, HSP70 and HSP27 were upregulated in all cell lines under HSP90 inhibition. Apoptosis was an early event in all three HSP90 inhibited cell lines and determined by CASPASE-3 and poly (ADP-ribose) polymerase assay. In contrast, gemcitabine treated cells did not show significant apoptosis. Conclusions: Functional inhibition of HSP90 disrupts multiple signalling cascades in gemcitabine and 5-FU resistant PC. Water-soluble 17DMAG is equally effective as 17AAG. The up-regulation of HSP70 and HSP27 indicates counter regulation of tumor cells to prevent apoptosis and should be addressed when considering HSP90 inhibition in PC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B86.

TRPV1 as an anti-ferroptotic target in osteoarthritis.

Defects in the proton-translocating NADH-quinone oxidoreductase (complex I) of mammalian mitochondria are linked to neurodegenerative disorders. The mechanism leading to cell death elicited by complex I deficiency remains elusive. We have shown that expression of a rotenone-insensitive yeast NADH-quinone oxidoreductase (Ndi1) can rescue mammalian cells from complex I dysfunction. By using the Ndi1 enzyme, we have investigated the key events in the process of cell death using a rat dopaminergic cell line, PC12. We found that complex I inhibition provokes the following events: 1) activation of specific kinase pathways; 2) release of mitochondrial proapoptotic factors, apoptosis inducing factor, and endonuclease G. AS601245, a kinase inhibitor, exhibited significant protection against these apoptotic events. The traditional caspase pathway does not seems to be involved because caspase 3 activation was not observed. Our data suggest that overproduction of reactive oxygen species (ROS) caused by complex I inhibition is responsible for triggering the kinase activation, for the release of the proapoptotic factors, and then for cell death. Nearly perfect prevention of apoptotic cell death by Ndi1 agrees with our earlier observation that the presence of Ndi1 diminishes rotenone-induced ROS generation from complex I. In fact, this study demonstrated that Ndi1 keeps the redox potential high even in the presence of rotenone. Under these conditions, ROS formation by complex I is known to be minimal. Possible use of our cellular model is discussed with regard to development of therapeutic strategies for neurodegenerative diseases caused by complex I defects.

Although both the heat shock protein 70 (HSP70) and the activating transcription factor 5 (ATF5) have been shown to promote cell survival of transformed cells but not survival of non-transformed cells, the relationship of the two molecules is unknown. Here we show that HSP70 and ATF5 are concomitantly up-regulated upon transient but down-regulated over prolonged cellular stress and apoptotic stimulation in the rat C6 glioma and human U87 glioma cells. HSP70 interacts strongly with the N-terminal activation domain of ATF5, which is expected to be rigid and uniquely structured under physiological conditions because of extraordinary high concentration (over 25%) of proline residues. Binding of HSP70 to ATF5 is an ATP-driven process and requires functional ATPase on the nucleotide binding domain of the HSP70 molecule. Overexpression of HSP70 dramatically stabilizes the ATF5 protein, which is otherwise subject to rapid degradation, facilitated by both proteasome-dependent and caspase-dependent processes, whereas HSP70 depletion leads to acceleration of ATF5 degradation and transcription repression of Bcl-2 and Egr-1, which are downstream targets of ATF5 in C6 and U87 glioma cells. Our data reveal an essential role for HSP70 in maintaining high levels of ATF5 expression in glioma cells and support the conclusion that ATF5 is an important substrate protein of HSP70 that mediates HSP70-promoted cell survival in glioma cells.

Heat shock protein 90 regulates necroptosis by modulating multiple signaling effectors.