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Glioblastoma (GBM) is the most frequently diagnosed primary central nervous system tumor in adults. Despite the standard of care therapy, which includes surgical resection, temozolomide chemotherapy, radiation and the newly added tumor-treating fields, median survival remains only ∼20 months. Unfortunately, GBM has a ∼100% recurrence rate, but after recurrence there are no Food and Drug Administration-approved therapies to limit tumor growth and enhance patient survival, as these tumors are resistant to temozolomide (TMZ). Recently, our laboratory reported that lucanthone slows GBM by inhibiting autophagic flux through lysosome targeting and decreases the number of Olig2+ glioma stem-like cells (GSC) in vitro and in vivo. We now additionally report that lucanthone efficiently abates stemness in patient-derived GSC and reduces tumor microtube formation in GSC, an emerging hallmark of treatment resistance in GBM. In glioma tumors derived from cells with acquired resistance to TMZ, lucanthone retains the ability to perturb tumor growth, inhibits autophagy by targeting lysosomes, and reduces Olig2 positivity. We also find that lucanthone may act as an inhibitor of palmitoyl protein thioesterase 1. Our results suggest that lucanthone may function as a potential treatment option for GBM tumors that are not amenable to TMZ treatment. SIGNIFICANCE STATEMENT: We report that the antischistosome agent lucanthone impedes tumor growth in a preclinical model of temozolomide-resistant glioblastoma and reduces the numbers of stem-like glioma cells. In addition, it acts as an autophagy inhibitor, and its mechanism of action may be via inhibition of palmitoyl protein thioesterase 1. As there are no defined therapies approved for recurrent, TMZ-resistant tumor, lucanthone could emerge as a treatment for glioblastoma tumors that may not be amenable to TMZ both in the newly diagnosed and recurrent settings., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Glioblastoma is the most common and aggressive primary brain tumor in adults. Median survival time remains at 16-20 months despite multimodal treatment with surgical resection, radiation, temozolomide and tumor-treating fields therapy. After genotoxic stress glioma cells initiate cytoprotective autophagy, which contributes to treatment resistance, limiting the efficacy of these therapies and providing an avenue for glioma recurrence. Antagonism of autophagy steps has recently gained attention as it may enhance the efficacy of classical chemotherapies and newer immune-stimulating therapies. The modulation of autophagy in the clinic is limited by the low potency of common autophagy inhibitors and the inability of newer ones to cross the blood-brain barrier. Herein, we leverage lucanthone, an anti-schistosomal agent which crosses the blood-brain barrier and was recently reported to act as an autophagy inhibitor in breast cancer cells. Our studies show that lucanthone was toxic to glioma cells by inhibiting autophagy. It enhanced anti-glioma temozolomide (TMZ) efficacy at sub-cytotoxic concentrations, and suppressed the growth of stem-like glioma cells and temozolomide-resistant glioma stem cells. In vivo lucanthone slowed tumor growth: reduced numbers of Olig2 + glioma cells, normalized tumor vasculature, and reduced tumor hypoxia. We propose that lucanthone may serve to perturb a mechanism of temozolomide resistance and allow for successful treatment of TMZ-resistant glioblastoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Radin, Smith, Moushiaveshi, Wolf, Bases and Tsirka.)

Deoxycytidine deaminase enzyme activity was reduced in lysates of human leukemic THP1 cells 24 h after transfection with siRNA designed to inhibit cell synthesis of heat shock protein 70 (Hsp70)1a and Hsp701b. The cytidine deaminase enzyme activity from the cell lysates was purified from an affinity column which contained bound single-stranded oligodeoxycytidylic acid. Deficient enzyme activity in certain elution fractions from the siRNA-transfected cells was restored by including recombinant HSP 70 in the assays. Enzyme activity in some other fractions was increased after siRNA transfection. Activation-induced cytidine deaminase (AID) is a central factor in the immune response. A more specific assay for AID was used to study the influence of Hsp70 on AID activity. Unlike Hsp70's ability to stimulate certain enzymes of DNA base excision repair and other cytidine deaminases, it had little effect on AID activity in vitro, or was weakly inhibitory.

Base excision repair (BER) of DNA damage in irradiated THP1 human leukemic cells was stimulated by pretreating the cells with exogenous recombinant Hsp70. The treatment of THP1 cells with recombinant Hsp70 in cell culture promoted repair by reducing the frequency of apurinic, apyrimidinic (AP) sites in DNA before and after 1.3 Gy of radiation. However, by 30 minutes after 2.6 Gy, accelerated repair of abasic sites supervened, which may contribute to the loss of the very-low-dose cell hypersensitivity seen in clonogenic studies of other laboratories. After irradiation with 2.6 Gy, the crucial initial glycosylase step was markedly incomplete when cells had been transfected 24 hours before with a small interfering RNA (siRNA) designed to inhibit synthesis of Hsp70. In confirmation, lysates from irradiated siRNA-treated cells after 2.6 Gy were deficient in uracil glycosylase activity (UDG). Transfection with a scrambled RNA of the same size did not interfere with the glycosylase step, ie, the prompt conversion of damaged pyrimidine sites to abasic sites as well as the subsequent repair of those sites. BER measured by reduction of DNA AP sites before and after low-dose radiation was also deficient in THP1 cells that had been transfected with the siRNA designed to inhibit synthesis of Hsp70. These results implicate BER and the participation of Hsp70 in the repair of DNA in human leukemic cells with the doses of ionizing radiation used in clinical regimens.

Pretreatment of human leukemia THP-1 cells with heat shock protein Hsp70 (Hsp70) protected them from the cell-lethal effects of the topoisomerase II inhibitor, lucanthone and from ionizing radiation. Cell viability was scored in clonogenic assays of single cells grown in liquid medium containing 0.5% methyl cellulose. Colonies were observed and rapidly scored after staining with the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide. The frequency of abasic sites in the deoxyribonucleic acid (DNA) of THP-1 cells was reduced when these cells were treated with Hsp70. Hsp70 is presumed to have protected the cells by promoting repair of cell DNA, in agreement with previous studies that showed that Hsp70 enhanced base excision repair by purified enzymes. The shoulders of radiation dose-response curves were enhanced by pretreatment of cells with Hsp70 and, importantly, were reduced when cells were transfected with ribonucleic acid designed to silence Hsp70. Hsp70 influenced repair of sublethal damage after radiation.

Base excision repair (BER) of damaged deoxyribonucleic acid (DNA) is a multistep process during which potentially lethal abasic sites temporarily exist. Repair of these lesions is greatly stimulated by heat shock protein 70 (Hsp70), which enhances strand incision and removal of the abasic sites by human apurinic-apyrimidinic endonuclease (HAP1). The resulting single-strand gaps must then be filled in. Here, we show that Hsp70 and its 48- and 43-kDa N-terminal domains greatly stimulated filling in the single-strand gaps by DNA polymerase beta, a novel finding that extends the role of Hsps in DNA repair. Incorporation of deoxyguanosine monophosphate (dGMP) to fill in single-strand gaps in DNA phagemid pBKS by DNA polymerase beta was stimulated by Hsp70. Truncated proteins derived from the C-terminus of Hsp70 as well as unrelated proteins were less effective, but proteins derived from the N-terminus of Hsp70 remained efficient stimulators of DNA polymerase beta repair of DNA single-strand gaps. In agreement with these results, repair of a gap in a 30-bp oligonucleotide by polymerase beta also was strongly stimulated by Hsp70 although not by a truncated protein from the C-terminus of Hsp70. Sealing of the repaired site in the oligonucleotide by human DNA ligase 1 was not specifically stimulated by Hsp-related proteins. Results presented here now implicate and extend the role of Hsp70 as a partner in the enzymatic repair of damaged DNA. The participation of Hsp70 jointly with base excision enzymes improves repair efficiency by mechanisms that are not yet understood.

We previously demonstrated the stimulation of human apurinic/apyrimidinic endonuclease 1 (HAP1) by heat shock protein 70 (HSP70). In this work, we further defined the functional interaction between these proteins. Digestion of HSP70 by trypsin released 48 and 43 kDa amino terminal fragments that retained the ability to stimulate HAP1. In agreement with this result, an HSP70 N-terminal deletion mutant protein containing amino acids 1-385 was comparable to the full-length protein in its ability to enhance HAP1 activity. HSP70 mutants containing carboxy terminal amino acids 386-640 stimulated HAP1 only slightly, as did unrelated proteins. These results implicate the amino terminal portion of HSP70 in stimulating the activity of HAP1.

The interaction of human heat shock protein 70 (HSP70) with human apurinic/apyrimidinic endonuclease (HAP1) was demonstrated by coimmunoprecipitation. A combination of HSP70 and HAP1 also caused a shift in the electrophoretic mobility of a DNA fragment containing an apurinic/apyrimidinic site. The functional consequence of the HSP70/HAP1 interaction was a 10-100-fold enhancement of endonuclease activity at abasic sites. The physical and functional interaction between HSP70 and HAP1 did not require the addition of ATP. The association of HSP70 and a key base excision repair enzyme suggests a role for heat shock proteins in promoting base excision repair. These findings provide a possible mechanism by which HSP70 protects cells against oxidative stress.

Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase beta (beta pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150-180-kDa fractions to 30- 70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein-protein associations are disrupted, but beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and beta pol in protein-protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150-180-kDa and 30-70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein-protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.

Purpose: To determine if lucanthone crossed the blood-brain barrier in experimental animals; and to determine accelerated tumor regression of human brain metastases treated jointly with lucanthone and whole brain radiation., Methods and Materials: The organ distribution of 3H lucanthone in mice and 125I lucanthone in rats was determined to learn if lucanthone crossed the blood-brain barrier. Size determinations were made of patients' brain metastases from magnetic resonance images or by computed tomography before and after treatment with 30 Gy whole brain radiation alone or with lucanthone., Results: The time course of lucanthone's distribution in brain was identical to that in muscle and heart after intraperitoneal or intravenous administration in experimental animals. Lucanthone, therefore, readily crossed the blood-brain barrier in experimental animals., Conclusion: Compared with radiation alone, the tumor regression in patients with brain metastases treated with lucanthone and radiation was accelerated, approaching significance using a permutation test at p = 0.0536.

It was demonstrated previously that a deoxyribophosphodiesterase (dRpase) activity is associated with the DNA repair enzyme exonuclease I, and that this activity is stimulated by the addition of the E. coli single-stranded DNA-binding protein (Ssb). This activity catalyzes the release of deoxyribose-phosphate groups at apurinic/apyrimidinic (AP) sites in the DNA that have been cleared by the action of an AP endonuclease. We have now used the yeast two-hybrid system to demonstrate that a protein-protein interaction occurs between exonuclease I and Ssb. When the E. coli ssb gene was fused in frame to the DNA-activating domain of the GAL4 transcriptional activator and the exonuclease I gene was fused in frame to the DNA-binding domain, a functional GAL4 transcriptional activator was produced as determined by growth of yeast on selective medium and the measurement of beta-galactosidase activity. We have also demonstrated that Ssb can stimulate the dRpase activity of exonuclease I using double-stranded bacteriophage M13 DNA containing several strand interruptions at incised AP sites. These results suggest that Ssb may be required for efficient base-excision repair in bacteria.

The time-course of repair of irradiated primate alpha DNA was studied after transfection and recovery from rat NRK cells. Rat cells were chosen for transfection because they have no alpha DNA. Plasmid pBUC4 alpha 10, containing 10 tandem 172 bp alpha DNA subunits in its 5 kbp DNA, was irradiated and introduced into the rat cells by electroporation. The transfected alpha DNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected alpha DNA in time-course experiments. alpha DNA continuously entered nuclei for processing in the first 2.5 h after transfection. The pool of damaged bases in alpha DNA in NRK nuclei was detectable 2.5 h after transfection. Radiation-induced alpha DNA fragments of electrophoretic mobility intermediate between those of unit nucleotide length were prominent in sequencing gel analyses of alpha DNA for 5-150 min after transfection. These intermediate mobility fragments initially disappeared with T 1/2 of 6-20 min. The alpha DNAs of intermediate mobility are presumed to be intermediates in DNA repair. Residual DNA base damage which had not been processed in the transfected cells could later be unmasked in vitro by conversion to strand breaks by beta-elimination using heat and piperidine or endonuclease III of E. coli. Irradiation of the recipient NRK cells with 5 Gy 4 hours before transfection prolonged the time during which intermediate mobility species could be found, consistent with the increased frequency of intermediate mobility species observed in DNA of monkey CV-1 cells pretreated with small doses of radiation before 300 Gy (Bases et al. 1990).

The alkylating mutagens N-methyl-N'-nitro-N-nitrosoguanidine, methyl methanesulfonate, and N-nitroso-methylurea induced immunoreactivity to antinucleoside antibodies in human peripheral blood lymphocytes in vitro. This could also be detected in lymphocytes taken from a patient soon after i.v. administration of cyclophosphamide. The immunoreactivity response, which indicates denatured DNA or DNA single-strand breaks, was scored by immunofluorescent or immunoperoxidase techniques. Examination of blood from 10 normal subjects showed that 32 +/- 4% (S.E.) of resting peripheral blood lymphocytes were immunoreactive to antinucleoside antibodies. We have shown that these naturally occurring immunoreactive lymphocytes are largely accounted for by a subpopulation of thymus-derived lymphocytes bearing the Fc receptor for immunoglobulin M. The presence of these cells did not interfere with the use of peripheral blood lymphocytes for in vitro measurement of additional immunoreactivity caused by alkylating mutagens. The response proved to be dose dependent; up to 90% of lymphocytes could be rendered immunoreactive. Parallel studies with HeLa cells showed a similar dose-response relationship between mutagen action and immunoreactivity. With some agents, the immunoreactivity technique detected effects at lower concentrations than could be detected by HeLa cell survival studies. With N-nitrosomethylurea, measurement of DNA repair synthesis by [3H]thymidine autoradiography showed that in HeLa cells these two parameters of response to DNA damage increased in parallel. Our results provide a new basis for detecting the action of alkylating mutagens on human lymphocytes in vitro or in vivo.

A fibroblast cell line (3T3) and normal rat peritoneal fluid cells were exposed in vitro to pulsed ultrasound from a diagnostic instrument (Smith-Kline "Ekoline 20"). We report here on ultrastructural changes in both cell types and on altered motility patterns in 3T3 fibroblasts. Abnormal motility was detectable 10 generations after exposure. X-irradiation and ultraviolet light elicited similar effects on cell motion. It is suggested that the cellular effects of diagnostic levels of ultrasound be further examined both in vitro and in vivo.

As part of its evaluation of the Clinical Cancer Education Program (CCEP) at the Albert Einstein College of Medicine, the Office of Educational Research and Evaluation analyzed student performance on neoplastic items of the National Board of Medical Examiners (NBME) Examination Part II. The evaluation provided a successful application of the American Association for Cancer Education (AACE) coding schema for analysis of neoplastic items. Two examinations were analyzed in order to determine the frequency with which specific disease sites, treatment modalities, and question emphasis items appeared. With reference to disease site, the greatest number of questions on both examinations dealt with gynecological issues while the fewest dealt with lung and hematologic-related disease. A breakdown of questions into various treatment modalities indicated that of items that could be categorized, most dealt with surgical treatment, while other therapeutic modalities were given little or no emphasis. Looking at question emphases, the most frequently asked questions referred to diagnostic tests and stratagems, whereas the least amount of emphasis was placed on rehabilitative and psychosocial aspects of the disease. These findings corroborate those reported by Ruckdeschel and his associates and point out several limitations to the content validity of the neoplastic items on NBME Part II.