ER-localized JmjC domain-containing protein JMJD8 targets STING to promote immune evasion and tumor growth in breast cancer

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Chaperone-rich cell lysates, immune activation and tumor vaccination

We have utilized a free-solution-isoelectric focusing technique (FS-IEF) to obtain chaperone-rich cell lysates (CRCL) fractions from clarified tumor homogenates. The FS-IEF technique for enriching multiple chaperones from tumor lysate is relatively easy and rapid, yielding sufficient immunogenic material for clinical use. We have shown that tumor-derived CRCL carry antigenic peptides. Dendritic cells (DCs) uptake CRCL and cross-present the chaperoned peptides to T cells. Tumor-derived CRCL induce protective immune responses against a diverse range of murine tumor types in different genetic backgrounds. When compared to purified heat shock protein 70 (HSP70), single antigenic peptide or unfractionated lysate, CRCL have superior ability to activate/mature DCs and are able to induce potent, long lasting and tumor specific T-cell-mediated immunity. While CRCL vaccines were effective as stand-alone therapies, the enhanced immunogenicity arising from CRCL-pulsed DC as a vaccine indicates that CRCL could be the antigen source of choice for DC-based anti-cancer immunotherapies. The nature of CRCL's enhanced immunogenicity may lie in the broader antigenic peptide repertoire as well as the superior immune activation capacity of CRCL. Exongenous CRCL also supply danger signals in the context of apoptotic tumor cells and enhance the immunogenicity of apoptotic tumor cells, leading to tumor-specific T cell dependent long-term immunity. Moreover, CRCL based vaccines can be effectively combined with chemotherapy to treat cancer. Our findings indicate that CRCL have prominent adjuvant effects and are effective sources of tumor antigens for pulsing DCs. Tumor-derived CRCL are promising anti-cancer vaccines that warrant clinical research and development.     

The ubiquitin E3 ligase TRIM10 promotes STING aggregation and activation in the Golgi apparatus

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Dysfunctional telomeres through mitostress‐induced cGAS/STING activation to aggravate immune senescence and viral pneumonia

Disproportionately high incidence and mortality of respiratory infection such as influenza A virus (IAV) and SARS‐CoV‐2 have been evidenced in the elderly, but the role and the mechanism of age‐associated immune deregulation in disease exacerbation are not well defined. Using a late generation of mice deficient in telomerase RNA (Terc^−/−), we herein demonstrated that aged mice were exquisitely susceptible to respiratory viral infection, with excessive inflammation and increased mortality. Furthermore, we identified the cGAS/STING pathway, which was essentially induced by the leaked mitochondrial DNA, as a biologically relevant mechanism contributing to exaggerated inflammation in Terc^−/− mice following viral infection. Innate immune cells, mainly, macrophages with shortened telomeres, exhibited hallmarks of cellular senescence, mitochondrial distress, and aberrant activation of STING and NLRP3 inflammasome pathways, which predisposed mice to severe viral pneumonia during commonly mild infections. Application of STING inhibitor and, more importantly, senolytic agent, reduced the burden of stressed macrophages, improved mitochondrial integrity, and suppressed STING activation, thereby conferring the protection for Terc^−/− mice against respiratory infection. Together, the findings expand our understanding of innate immune senescence and reveal the potential of the senolytics as a promising treatment to alleviate the symptom of viral pneumonia, particularly for the older population.     

Elimination of tumor by CD47/PD-L1 dual-targeting fusion protein that engages innate and adaptive immune responses

The host immune system generally serves as a barrier against tumor formation. Programmed death-ligand 1 (PD-L1) is a critical “don't find me” signal to the adaptive immune system, whereas CD47 transmits an anti-phagocytic signal, known as the “don't eat me” signal, to the innate immune system. These and similar immune checkpoints are often overexpressed on human tumors. Thus, dual targeting both innate and adaptive immune checkpoints would likely maximize anti-tumor therapeutic effect and elicit more durable responses. Herein, based on the variable region of atezolizumab and consensus variant 1 (CV1) monomer, we constructed a dual-targeting fusion protein targeting both CD47 and PD-L1 using “Knobs-into-holes” technology, denoted as IAB. It was effective in inducing phagocytosis of tumor cells, stimulating T-cell activation and mediating antibody-dependent cell-mediated cytotoxicity in vitro. No obvious sign of hematological toxicity was observed in mice administered IAB at a dose of 100 mg/kg, and IAB exhibited potent antitumor activity in an immune-competent mouse model of MC38. Additionally, the anti-tumor effect of IAB was impaired by anti-CD8 antibody or clodronate liposomes, which implied that both CD8+ T cells and macrophages were required for the anti-tumor efficacy of IAB and IAB plays an essential role in the engagement of innate and adaptive immune responses. Collectively, these results demonstrate the capacity of an elicited endogenous immune response against tumors and elucidate essential characteristics of synergistic innate and adaptive immune response, and indicate dual blockade of CD47 and PD-L1 by IAB may be a synergistic therapy that activates both innate and adaptive immune response against tumors.     

PH domain of ELMO functions in trans to regulate Rac activation via Dock180

The members of the Dock180 superfamily of proteins are novel guanine nucleotide exchange factors (GEF) for Rho family GTPases and are linked to multiple biological processes from worms to mammals. ELMO is a critical regulator of Dock180, and the Dock180-ELMO complex functions as a bipartite GEF for Rac. We identified a mechanism wherein the PH domain of ELMO, by binding the Dock180-Rac complex in trans, stabilizes Rac in the nucleotide-free transition state. Mutagenesis studies reveal that this ELMO PH domain-dependent regulation is essential for the Dock180-ELMO complex to function in phagocytosis and cell migration. Genetic rescue studies in Caenorhabditis elegans using ELMO and its homolog CED-12 support the above observations in vivo. These data reveal a new mode of action of PH domains and a novel, evolutionarily conserved mechanism by which a bipartite GEF can activate Rac.     

Lactate stimulates vasculogenic stem cells via the thioredoxin system and engages an autocrine activation loop involving hypoxia-inducible factor 1

The recruitment and differentiation of circulating stem/progenitor cells (SPCs) in subcutaneous Matrigel in mice was assessed. There were over one million CD34⁺ SPCs per Matrigel plug 18 h after Matrigel implantation, and including a polymer to elevate the lactate concentration increased the number of SPCs by 3.6-fold. Intricate CD34⁺ cell-lined channels were linked to the systemic circulation, and lactate accelerated cell differentiation as evaluated based on surface marker expression and cell cycle entry. CD34⁺ SPCs from lactate-supplemented Matrigel exhibited significantly higher concentrations of thioredoxin 1 (Trx1) and hypoxia-inducible factor 1 (HIF-1) than cells from unsupplemented Matrigel, whereas Trx1 and HIF-1 in CD45⁺ leukocytes were not elevated by lactate. Results obtained using small inhibitory RNA (siRNA) specific to HIF-1 and mice with conditionally HIF-1 null myeloid cells indicated that SPC recruitment and lactate-mediated effects were dependent on HIF-1. Cells from lactate-supplemented Matrigel had higher concentrations of phosphorylated extracellular signal-regulated kinases 1 and 2, Trx1, Trx reductase (TrxR), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) than cells from unsupplemented Matrigel. SPC recruitment and protein changes were inhibited by siRNA specific to lactate dehydrogenase, TrxR, or HIF-1 and by oxamate, apocynin, U0126, N-acetylcysteine, dithioerythritol, and antibodies to VEGF or SDF-1. Oxidative stress from lactate metabolism by SPCs accelerated further SPC recruitment and differentiation through Trx1-mediated elevations in HIF-1 levels and the subsequent synthesis of HIF-1-dependent growth factors.     

PI3Kγ contributes to MEK1/2 activation in oxidative glutamate toxicity via PDK1

The role of phosphoinositide 3‐kinase (PI3K) in oxidative glutamate toxicity is not clear. Here, we investigate its role in HT22 mouse hippocampal cells and primary cortical neuronal cultures, showing that inhibitors of PI3K, LY294002, and wortmannin suppress extracellular hydrogen peroxide (H₂O₂) generation and increase cell survival during glutamate toxicity in HT22 cells. The mitogen‐activated protein kinase kinase (MEK) inhibitor U0126 also reduced glutamate‐induced H₂O₂ generation and inhibited phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2. LY294002 was seen to abolish phosphorylation of both ERK1/2 and Akt. A small interfering RNA (siRNA) study showed that PI3Kβ and PI3Kγ, rather than PI3Kα and PI3Kδ, contribute to glutamate‐induced H₂O₂ generation and cell death. PI3Kγ knockdown also inhibited glutamate‐induced ERK1/2 phosphorylation, whereas transfection with the constitutively active form of human PI3Kγ (PI3Kγ‐CAAX) triggered MEK1/2 and ERK1/2 phosphorylation and H₂O₂ generation without glutamate exposure. This H₂O₂ generation was reduced by inhibition of MEK. Transfection with kinase‐dead 3‐phosphoinositide‐dependent protein kinase 1 (PDK1‐KD) reduced glutamate‐induced ERK1/2 phosphorylation and H₂O₂ generation. Accordingly, cotransfection of cells with PDK1‐KD and PI3Kγ‐CAAX suppressed PI3Kγ‐CAAX‐triggered ERK1/2 phosphorylation and H₂O₂ generation. These results suggest that activation of PI3Kγ induces ERK1/2 phosphorylation, leading to extracellular H₂O₂ generation via PDK1 in oxidative glutamate toxicity.

     

Mirtazapine inhibits tumor growth via immune response and serotonergic system

To study the tumor inhibition effect of mirtazapine, a drug for patients with depression, CT26/luc colon carcinoma-bearing animal model was used. BALB/c mice were randomly divided into six groups: two groups without tumors, i.e. wild-type (no drug) and drug (mirtazapine), and four groups with tumors, i.e. never (no drug), always (pre-drug, i.e. drug treatment before tumor inoculation and throughout the experiment), concurrent (simultaneously tumor inoculation and drug treatment throughout the experiment), and after (post-drug, i.e. drug treatment after tumor inoculation and throughout the experiment). The "psychiatric" conditions of mice were observed from the immobility time with tail suspension and spontaneous motor activity post tumor inoculation. Significant increase of serum interleukin-12 (sIL-12) and the inhibition of tumor growth were found in mirtazapine-treated mice (always, concurrent, and after) as compared with that of never. In addition, interferon-γ level and immunocompetent infiltrating CD4+/CD8+ T cells in the tumors of mirtazapine-treated, tumor-bearing mice were significantly higher as compared with that of never. Tumor necrosis factor-α (TNF-α) expressions, on the contrary, are decreased in the mirtazapine-treated, tumor-bearing mice as compared with that of never. Ex vivo autoradiography with [(123)I]ADAM, a radiopharmaceutical for serotonin transporter, also confirms the similar results. Notably, better survival rates and intervals were also found in mirtazapine-treated mice. These findings, however, were not observed in the immunodeficient mice. Our results suggest that tumor growth inhibition by mirtazapine in CT26/luc colon carcinoma-bearing mice may be due to the alteration of the tumor microenvironment, which involves the activation of the immune response and the recovery of serotonin level.     

Phosphatidylinositol-3 kinase dependent MAP kinase activation via p21ras in endothelial cells exposed to cyclic strain

Hemodynamic forces play a key role in the modulation of the morphology and function of the endothelium by activating several kinases. We have previously shown that cyclic strain, a repetitive mechanical stretch, induces activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), members of the mitogen activated protein (MAP) kinase family. In order to investigate the upstream pathway of strain-induced ERK1/2 activation, we examined p21ras activation by cyclic strain and the effect of wortmannin and LY294002, phosphatidylinositol-3 kinase (PI 3-kinase) inhibitors on ERK1/2 phosphorylation. Cyclic strain induced a transient and rapid activation of p21ras at 1 min after strain. Wortmannin inhibited strain-induced ERK1/2 activation by 56.3 and 86.3 %, respectively. LY294002 inhibited ERK1 activation completely and ERK2 activation by 42.9%. These results suggest a possible involvement of p21ras and PI 3-kinase in the signal transduction pathway leading to the strain-induced ERK1/2 activation.     

MITA/STING: A central and multifaceted mediator in innate immune response

The recognition of nucleic acids is a general strategy used by the host to detect invading pathogens. Many studies have established that MITA/STING is a central component in the innate immune response to cytosolic DNA and RNA derived from pathogens. MITA can act both as a direct sensor of cyclic dinucleotides (CDNs) and as an adaptor for the recruitment of downstream signaling components. In both roles, MITA is part of signaling cascades that orchestrate innate immune defenses against various pathogens, including viruses, bacteria and parasites. Here, we highlight recent studies that have uncovered the molecular mechanisms of MITA-mediated signal transduction and regulation, and discuss some notable issues that remain elusive.     

Vulnerability to glutamate toxicity of dopaminergic neurons is dependent on endogenous dopamine and MAPK activation

Dopaminergic neurons are more vulnerable than other types of neurons in cases of Parkinson disease and ischemic brain disease. An increasing amount of evidence suggests that endogenous dopamine plays a role in the vulnerability of dopaminergic neurons. Although glutamate toxicity contributes to the pathogenesis of these disorders, the sensitivity of dopaminergic neurons to glutamate toxicity has not been clarified. In this study, we demonstrated that dopaminergic neurons were preferentially affected by glutamate toxicity in rat mesencephalic cultures. Glutamate toxicity in dopaminergic neurons was blocked by inhibiting extracellular signal-regulated kinase (ERK), c- jun N-terminal kinase, and p38 MAPK. Furthermore, depletion of dopamine by α-methyl- dl - p -tyrosine methyl ester (α-MT), an inhibitor of tyrosine hydroxylase (TH), protected dopaminergic neurons from the neurotoxicity. Exposure to glutamate facilitated phosphoryration of TH at Ser31 by ERK, which contributes to the increased TH activity. Inhibition of ERK had no additive effect on the protection offered by α-MT, whereas α-MT and c- jun N-terminal kinase or p38 MAPK inhibitors had additive effects and yielded full protection. These data suggest that endogenous dopamine is responsible for the vulnerability to glutamate toxicity of dopaminergic neurons and one of the mechanisms may be an enhancement of dopamine synthesis mediated by ERK.     

Paradoxical effects of cytokines in tumor immune surveillance and tumor immune escape

The role of cytokines in modulating the formation of new tumors is mediated by their ability to regulate antigen-specific anti-tumor responses and by the activation of non-specific mechanisms, including those involved in the processes of inflammation and innate resistance. Cytokines may influence the growth of tumors by acting directly on tumor cells as growth promoting or growth inhibiting factors or indirectly by attracting inflammatory cell types and affecting angiogenesis. Due to the potency and complexity of cytokine activity against tumor growth, the improvement of cloning techniques and the availability of recombinant forms of different cytokines, a great effort has been made in the recent years to exploit this anti-tumor potential for cancer therapy. This important goal has been difficult to achieve in most cases due to toxicity of most cytokines which could not be dissociated from their anti-tumoral functions. Nevertheless, if well designed, treatment protocols and/or modifications of the cytokine molecules may in some situations augment the anti-tumor effects while limiting the toxicity. One of these molecular approaches could be the design of peptides containing the functional domain of certain cytokines, exemplified by IT9302, a peptide homologous to the functional domain of IL-10, which has demonstrated to increase tumor NK cell sensitivity.     

The chemokine CCL6 promotes innate immunity via immune cell activation and recruitment

Septic syndrome is a consequence of innate immune failure. Recent studies showed that the CC chemokine CCL6 enhanced antimicrobial immunity during experimental sepsis through an unknown mechanism. The present study demonstrates that transgenic CCL6 expression abolishes mortality in a septic peritonitis model via the modulation of resident peritoneal cell activation and, more importantly, through the recruitment of IFN-producing NK cells and killer dendritic cells into the peritoneum. Thus, CCL6 attenuates the immune failure during sepsis, in part, through a protective type 1-cytokine mediated mechanism.     

Superantigen-based tumor therapy: in vivo activation of cytotoxic T cells

We have recently demonstrated that the superantigen staphylococcal enterotoxin A (SEA) targets in vitro activated cytotoxic T lymphocytes against tumor cells expressing major histocompatibility complex (MHC) class II antigens. In this report we analyze the use of SEA as an immunoactivator in vivo. Treatment of mice with SEA activated a fraction of CD3⁺ T cells apparently as a function of their T cell receptor V expression. SEA induced interleukin-2 receptor expression and proliferation in both CD4⁺ and CD8⁺ T cells. This proliferative response was dose-dependent (0.1 – 100 µg/mouse), peaked during day 1 after treatment and declined to background levels within 4 days. The cytotoxic response, measured as cytotoxicity to SEA-coated MHC class II⁺ target cells (staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity, SDCC), was maximal at a dosage of 1 µg SEA/mouse. The SDCC was confined to the CD8⁺ T cell compartment, peaked 2 days after treatment and declined to background levels within 4 days. A second injection of SEA on day 5 after the first SEA treatment resulted in SDCC function with kinetics and magnitude identical to that seen after one injection. These results pave the way for the use of SEA in the treatment of MHC class II⁺ tumors.     

DNA immunization with HIV-1 tat mutated in the trans activation domain induces humoral and cellular immune responses against wild-type Tat

Intramuscular immunization of mice with plasmids encoding two transdominant negative mutants of the HIV-1 Tat protein (Tat22 and Tat22/37) elicited a humoral response to wild-type Tat that is comparable to that induced by inoculation of wild-type tat DNA or Tat protein. The percentage of the responders and the Ab titers continued to increase after three additional DNA boosts and pretreatment with bupivacaine at the site of inoculation, without a significant difference (p > 0.05) among the three groups of mice immunized with mutant and wild-type tat genes. By utilizing synthetic peptides representing the amino acid sequence of Tat, one major B cell epitope was defined within the cysteine-rich domain of Tat. Anti-Tat IgG Abs directed against this epitope were found in mice immunized with all tat DNA constructs, whereas different Tat epitopes were detected in mice immunized with the Tat protein. Similarly, IgG2a was the predominant isotype in DNA-immunized mice, with both mutants and wild-type tat genes, as compared with protein immunization, which induced mostly IgG1 and IgG3. Sera from most immunized mice neutralized the effect of extracellular Tat in activating HIV-1 replication. A cellular response was also elicited as indicated by the proliferation of splenocytes when stimulated with wild-type Tat. These results indicate that the wild-type Tat Ag is recognized by Abs and T cells induced by DNA immunization with mutated tat genes, suggesting the possible use of these Tat transdominant mutants, lacking viral trans activation activity and capable of blocking wild-type Tat activity, in the development of an anti-HIV-1 vaccine.     

Innate immune activation of CD4 T cells in salmonella-infected mice is dependent on IL-18

Production of IFN-gamma by CD4 T cells is generally thought to be mediated by TCR triggering, however, Ag-nonspecific activation of effector CD8 T cells has been reported in infection models. In this study, we demonstrate that Ag-experienced CD4 T cells in the spleen of Salmonella-infected mice acquire the capacity to rapidly secrete IFN-gamma in response to stimulation with bacterial lysate or LPS. This innate responsiveness of T cells was transient and most apparent during, and immediately following, active Salmonella infection. Furthermore, innate T cell production of IFN-gamma in response to bacterial lysate or LPS was Ag independent and could be induced in Listeria-infected mice and in the absence of MHC class II expression. IL-18 was required for maximal innate responsiveness of CD4 T cells in Salmonella-infected mice and for optimal bacterial clearance in vivo. These data demonstrate that CD4 T cells acquire the capacity to respond to innate stimuli during active bacterial infection, a process that may contribute significantly to amplifying effector responses in vivo.     

Thiazolides promote apoptosis in colorectal tumor cells via MAP kinase-induced Bim and Puma activation

While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance mechanisms in the tumor cells prevent cell death induction. In particular enzymes of the glutathion-S-transferase (GST) family represent a well-known detoxification mechanism, which limit the effect of chemotherapeutic drugs in tumor cells. Specifically, GST of the class P1 (GSTP1-1) is overexpressed in colorectal tumor cells and renders them resistant to various drugs. Thus, GSTP1-1 has become an important therapeutic target. We have recently shown that thiazolides, a novel class of anti-infectious drugs, induce apoptosis in colorectal tumor cells in a GSTP1-1-dependent manner, thereby bypassing this GSTP1-1-mediated drug resistance. In this study we investigated in detail the underlying mechanism of thiazolide-induced apoptosis induction in colorectal tumor cells. Thiazolides induce the activation of p38 and Jun kinase, which is required for thiazolide-induced cell death. Activation of these MAP kinases results in increased expression of the pro-apoptotic Bcl-2 homologs Bim and Puma, which inducibly bind and sequester Mcl-1 and Bcl-x_L leading to the induction of the mitochondrial apoptosis pathway. Of interest, while an increase in intracellular glutathione levels resulted in increased resistance to cisplatin, it sensitized colorectal tumor cells to thiazolide-induced apoptosis by promoting increased Jun kinase activation and Bim induction. Thus, thiazolides may represent an interesting novel class of anti-tumor agents by specifically targeting tumor resistance mechanisms, such as GSTP1-1.Glutathione-S-transferases (GSTs) represent a superfamily of cellular phase II detoxification enzyme. Specifically, GSTs catalyze the conjugation of electrophilic substances to the tripeptid glutathione (GSH, γ-L-glutamyl-L-cysteinylglycine). Thereby, hazardous metabolic products, xenobiotics and oxidative stress products become rapidly neutralized by GSTs, protecting cells from potentially damaging substances and carcinogens. Consequently, GSTs have a critical role in the detoxification of cells and inactivation of drugs.^{1, 2} At the present, seven classes of mammalian cytosolic GSTs are known, whose expression is regulated in a tissue-specific manner^{3, 4, 5} pointing toward a defined role of individual GSTs in the biotransformation of drugs and reactive compounds in diverse tissues.^{6, 7}GSTs have a critical role in tumor therapy, as numerous tumors overexpress various GSTs, which contribute to the development of resistance to chemotherapeutic treatments.^{8, 9} In particular, high expression levels of GST class pi (GSTP1-1) have been reported in a wide range of solid tumors, such as colon, breast, kidney, pancreas, lung, and ovarian cancer cells, and lymphoma.^{10, 11, 12} The sensitivity of these tumors toward chemotherapeutic drugs, such as cisplatin, doxorubicin, and etoposide, is negatively affected by high expression levels of GSTP1-1.^{13, 14, 15, 16, 17} Thus, overexpression of GSTP1-1 in solid tumors limits the therapeutic effects of different chemotherapeutic drugs via their GSTP1-1-mediated inactivation.This observation identifies GSTs in general and GSTP1-1 in particular as important therapeutic targets in the treatment of solid tumors. Consequently, small molecular inhibitors of GSTs have been developed in the past to modulate GST activities and drug resistance of tumor cells, thereby sensitizing tumor cells to anticancer drugs. The therapeutic effect of the competitive inhibitors ethacrynic acid (EA) was proven in a clinical trial;¹⁸ however, long-term utility of EA was limited by its strong diuretic properties.¹⁹ A somewhat different approach includes the GST-activated pro-drugs and the GSH analog TLK199. TLK199 is metabolized and subsequently inhibits GST activities, making it a more selective GST inhibitor.²⁰ However, thus far experimental and clinical data on solid tumors are missing.Thiazolides are a novel class of anti-infectious drugs used for the treatment of intestinal infection, and show a broad activity against intestinal pathogens.^{21, 22, 23, 24} The parent compound nitazoxanide (NTZ; 2-(acetolyloxy)-N-(5-nitro-2-thiazolyl)benzamide) is successfully used for the treatment of Giardia lamblia and Cryptosporidium parvum infections.^{25, 26, 27, 28} Though thiazolides generally have minimal side effects on host tissue cells during therapeutic treatments,²⁹ it was recently noticed that they promote apoptosis induction in colorectal tumor cells, however, sparing non-transformed cells.³⁰ Of interest, while the bromo-thiazolide RM4819 (N-(5-bromothiazol-2-yl)2-hydroxy-3-methylbenzamide) shows only reduced anti-microbial activity, both NTZ and RM4819 promote cell death in colorectal tumor cells. This indicates that the therapeutic targets of thiazolides are substantially different in intestinal parasites and colorectal tumor cells. Subsequent studies identified GSTP1-1 as a major RM4819-binding partner in colorectal tumor cells.³⁰ While it was initially thought that thiazolides are inhibitors of GSTP1-1, it is presently accepted that GSTP1-1 is required for thiazolide-induced cell death induction. Interestingly, an N-acetyl-L-cysteine (NAC)-induced increase in cellular GSH levels enhanced thiazolide-induced cell death, whereas it lowered the sensitivity toward chemotherapeutic drugs by promoting their GSTP1-1-mediated inactivation.³¹ Thus, thiazolides appear to represent a novel class of GSTP1-1-activated pro-drugs, activated likely by conjugation to GSH, rather than GSTP1-1 inhibitors. This makes thiazolides an interesting novel class of anti-tumor drugs specifically targeting tumors with elevated levels of GSTs, and GSTP1-1 an Achilles'' heel for the potential therapeutic action of thiazolides. While thiazolides alone are relatively slow and weak inducers of apoptosis in colorectal tumor cells, they profoundly synergize with inducers of the intrinsic apoptosis pathway, such as chemotherapeutic drugs, as well triggers of the extrinsic pathway, such as TRAIL (TNF-related apoptosis-inducing ligand).³¹The mechanism of thiazolide-induced apoptosis and sensitization of tumor cells to other apoptosis triggers is presently incompletely understood, although GSTP1-1, the activation of the MAP kinases, and the Bcl-2-regulated mitochondrial apoptosis pathways appear to have a critical role in this process.³¹ In this study we investigated in more detail the underlying molecular signaling pathways leading to thiazolide-induced cell death in colorectal tumor cells. We find that activity of both the MAP kinases p38 and Jun kinase (JNK) is critical for mediating thiazolide-induced apoptosis, as their combined inhibition blocks cell death induction. In particular JNK was found to be important for the induction and activation of the downstream effectors of the Bcl-2 family, that is, the BH3-only proteins Bim and Puma. Bim and Puma appear to activate the mitochondrial pathway by interacting with and neutralizing the anti-apoptotic Bcl-2 homolog Bcl-x_L, and inhibition of JNK prevented Bim and Puma induction, interaction with Bcl-x_L, and induction of apoptosis. Furthermore, thiazolides induced interaction of Bim with Mcl-1 and promote the degradation of Mcl-1. While an increase in cellular GSH levels inhibited chemotherapy-induced apoptosis, it resulted in a more robust activation of JNK, Bim induction, Mcl-1 degradation, and associated thiazolide-induced cell death.In summary, we here show that thiazolides are a novel group of GSTP1-1-activated pro-drugs, which activate the mitochondrial apoptosis pathway at different levels. Given that GSTs are highly overexpressed in numerous tumors and that GSTs contribute to therapy resistance of these tumors, thiazolides may become an interesting therapeutic option for the treatment of chemoresistant tumor cells.     

Caspase-3 dependent cleavage and activation of skeletal muscle phosphorylase b kinase

Phosphorylase b kinase (PhK) is a key enzyme involved in the conversion of glycogen to glucose in skeletal muscle and ultimately an increase in intracellular ATP. Since apoptosis is an ATP-dependent event, we investigated the regulation of skeletal muscle PhK during apoptosis. Incubation of PhK with purified caspase-3 in vitro resulted in the highly selective cleavage of the regulatory α subunit and resulted in a 2-fold increase in PhK activity. Edman protein sequencing of a stable 72 kD amino-terminal fragment and a 66 kD carboxy-terminal fragment revealed a specific caspase-3 cleavage site within the α subunit at residue 646 (DWMD↓G). Treatment of differentiated C2C12 mouse muscle myoblasts with the inducers of apoptosis staurosporine, TPEN, doxorubicin, or UV irradiation resulted in the disappearance of the α subunit of PhK as determined by immunoblotting, as well as a concurrent increase in caspase-3 activity. Moreover, induction of apoptosis by TPEN resulted in increased phosphorylase activity and sustained ATP levels throughout a 7 h time course. However, induction of apoptosis with staurosporine, also a potent PhK inhibitor, led to a rapid loss in phosphorylase activity and intracellular ATP, suggesting that PhK inhibition by staurosporine impairs the ability of apoptotic muscle cells to generate ATP. Thus, these studies indicate that PhK may be a substrate for caspase regulation during apoptosis and suggest that activation of this enzyme may be important for the generation of ATP during programmed cell death.