A significant problem affecting gene therapy approaches aiming at achieving long-term transgene expression is the immune response against the protein product of the therapeutic gene, which can reduce ...or eliminate the therapeutic effect. The problem is further exacerbated when therapy involves targeting an immunogenic tissue and/or one with a pre-existing inflammatory phenotype, such as dystrophic muscles. In this proof-of-principle study, we co-expressed a model antigen, bacterial β-galactosidase, with an immunosuppressive factor, indoleamine 2,3-dioxygenase 1 (IDO1), in muscles of the mdx mouse model of Duchenne muscular dystrophy. This treatment prevented loss of expression of the transgene concomitant with significantly elevated expression of T-regulatory (Treg) markers in the IDO1-expressing muscles. Moreover, co-expression of IDO1 resulted in reduced serum levels of anti-β-gal antibodies. These data indicate that co-expression of genes encoding immunomodulatory enzymes controlling kynurenine pathways provide a viable strategy for preventing loss of transgenes targeted into dystrophic muscles with pre-existing inflammation.
Brody disease (BD, OMIM #601003) is an inherited skeletal muscle disease clinically characterized by exercise-induced impairment of muscle relaxation and stiffness due to the delay in the Ca2+ ...re-uptake in the sarcoplasmic reticulum (SR)1, 2. The delayed muscle relaxation mainly affects legs, harms, hands and eyelids and usually improves after a few minutes rest 1-3. Patients frequently report myalgia, painless or mildly painful cramps whereas recurrent rhabdomyolysis have been described in a few cases 2, 3. A reduction in sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity characterizes the skeletal muscle of these patients 2-5. This article is protected by copyright. All rights reserved.
Photodynamic therapy is a promising antitumor treatment modality approved for the management of both early and advanced tumors. The mechanisms of its antitumor action include generation of singlet ...oxygen and reactive oxygen species that directly damage tumor cells and tumor vasculature. A number of mechanisms seem to be involved in the protective responses to PDT that include activation of transcription factors, heat shock proteins, antioxidant enzymes and antiapoptotic pathways. Elucidation of these mechanisms might result in the design of more effective combination strategies to improve the antitumor efficacy of PDT. Using DNA microarray analysis to identify stress-related genes induced by Photofrin-mediated PDT in colon adenocarcinoma C-26 cells, we observed a marked induction of heme oxygenase-1 (HO-1). Induction of HO-1 with hemin or stable transfection of C-26 with a plasmid vector encoding HO-1 increased resistance of tumor cells to PDT-mediated cytotoxicity. On the other hand, zinc (II) protoporphyrin IX, an HO-1 inhibitor, markedly augmented PDT-mediated cytotoxicity towards C-26 and human ovarian carcinoma MDAH2774 cells. Neither bilirubin, biliverdin nor carbon monoxide, direct products of HO-1 catalysed heme degradation, was responsible for cytoprotection. Importantly, desferrioxamine, a potent iron chelator significantly potentiated cytotoxic effects of PDT. Altogether our results indicate that HO-1 is involved in an important protective mechanism against PDT-mediated phototoxicity and administration of HO-1 inhibitors might be an effective way to potentiate antitumor effectiveness of PDT.
Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER)-resident chaperone and a major regulator of the unfolded protein response (UPR). Accumulating evidence indicate that GRP78 is ...overexpressed in many cancer cell lines, and contributes to the invasion and metastasis in many human tumors. Besides, GRP78 upregulation is detected in response to different ER stress-inducing anticancer therapies, including photodynamic therapy (PDT). This study demonstrates that GRP78 mRNA and protein levels are elevated in response to PDT in various cancer cell lines. Stable overexpression of GRP78 confers resistance to PDT substantiating its cytoprotective role. Moreover, GRP78-targeting subtilase cytotoxin catalytic subunit fused with epidermal growth factor (EGF-SubA) sensitizes various cancer cells to Photofrin-mediated PDT. The combination treatment is cytotoxic to apoptosis-competent SW-900 lung cancer cells, as well as to Bax-deficient and apoptosis-resistant DU-145 prostate cancer cells. In these cells, PDT and EGF-SubA cytotoxin induce protein kinase R-like ER kinase and inositol-requiring enzyme 1 branches of UPR and also increase the level of C/EBP (CCAAT/enhancer-binding protein) homologous protein, an ER stress-associated apoptosis-promoting transcription factor. Although some apoptotic events such as disruption of mitochondrial membrane and caspase activation are detected after PDT, there is no phosphatidylserine plasma membrane externalization or DNA fragmentation, suggesting that in DU-145 cells the late apoptotic events are missing. Moreover, in SW-900 cells, EGF-SubA cytotoxin potentiates PDT-mediated cell death but attenuates PDT-induced apoptosis. In addition, the cell death cannot be reversed by caspase inhibitor z-VAD, confirming that apoptosis is not a major cell death mode triggered by the combination therapy. Moreover, no typical features of necrotic or autophagic cell death are recognized. Instead, an extensive cellular vacuolation of ER origin is observed. Altogether, these findings indicate that PDT and GRP78-targeting cytotoxin treatment can efficiently kill cancer cells independent on their apoptotic competence and triggers an atypical, non-apoptotic cell death.
Photodynamic therapy (PDT) is a clinically approved and rapidly developing cancer treatment regimen. It is a minimally invasive two-stage procedure that requires administration of a photosensitizing ...agent followed by illumination of the tumor with visible light usually generated by laser sources. A third component of PDT is molecular oxygen which is required for the most effective antitumor effects. In the presence of the latter, light of an appropriate wavelength excites the photosensitizer thereby producing cytotoxic intermediates that damage cellular structures. PDT has been approved in many countries for the treatment of lung, esophageal, bladder, skin and head and neck cancers. The antitumor effects of this treatment result from the combination of direct tumor cell photodamage, destruction of tumor vasculature and activation of an immune response. The mechanisms of the direct photodamage of tumor cells, the signaling pathways that lead to apoptosis or survival of sublethaly damaged cells, and potential novel strategies of improving the antitumor efficacy of PDT are discussed.
Abstract
Mixed Lineage Kinase 4 (MLK4) is a serine/threonine kinase that plays a role in a variety of cellular processes, including migration, apoptosis and proliferation. It belongs to the family of ...MAP3 kinases, which regulate the activity of specific MAPKs (mitogen-activated protein kinases). MLK4 is the least described member of the family and it was proved to act as a functional MAP3 kinase that activates the JNK, ERK and NFκB pathways. The rationale of this project is based on the recent data showing gene amplification and mRNA upregulation of MLK4 in invasive breast carcinoma at the frequency of 23%. Here, we performed a series of experiments aiming to characterize the role of MLK4 in breast cancer development and progression. We started from identification of breast cancer cell lines that express high endogenous level of MLK4. Since clinical data indicated that MLK4 expression was higher in triple-negative breast cancer (TNBC), comparing to other breast cancer subtypes, we decided to further study the role of MLK4 in TNBC. We have generated cell lines with doxycycline-inducible knock-down of MLK4, HCC1806 and HCC1599, and we found that MLK4 depletion resulted in reduced cell proliferation. We also observed that the knock-down of MLK4 in these conditions caused the reduction in size of the spheroids. Moreover, we noticed that when MLK4 is depleted in HCC1806 cells, the migration and invasion was significantly impaired. Lastly, our results of immunohistochemistry in samples from breast cancer patients showed that higher levels of MLK4 in TNBC samples significantly correlated with the occurrence of lymph node metastasis. Currently, we are investigating molecular pathways such as JNK, MEK/ERK and NF-κB, that might be affected by knock-down of MLK4. Collectively, our findings indicate that high levels of MLK4 promote aggressive phenotype of breast cancer cells, by increasing their migratory and invasive potential.
Citation Format: Prelowska MK, Mehlich D, Lazniewski M, Kaminska K, Gorczynski AS, Korwat A, Sokolowska O, Golab J, Biernat W, Plewczynski D, Brognard J, Nowis D, Marusiak AA. The role of MLK4 amplification in progression of triple negative breast cancer abstract. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-09-07.
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