Lucanthone, a Potential PPT1 Inhibitor, Perturbs Stemness, Reduces Tumor Microtube Formation, and Slows the Growth of Temozolomide-Resistant Gliomas In Vivo.

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.

Lucanthone, Autophagy Inhibitor, Enhances the Apoptotic Effects of TRAIL through miR-216a-5p-Mediated DR5 Upregulation and DUB3-Mediated Mcl-1 Downregulation.

A lucanthone, one of the family of thioxanthenones, has been reported for its inhibitory effects of apurinic endonuclease-1 and autophagy. In this study, we investigated whether lucanthone could enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in various cancer cells. Combined treatment with lucanthone and TRAIL significantly induced apoptosis in human renal carcinoma (Caki and ACHN), prostate carcinoma (PC3), and lung carcinoma (A549) cells. However, combined treatment did not induce apoptosis in normal mouse kidney cells (TCMK-1) and normal human skin fibroblast (HSF). Lucanthone downregulated protein expression of deubiquitinase DUB3, and a decreased expression level of DUB3 markedly led to enhance TRAIL-induced apoptosis. Ectopic expression of DUB3 inhibited combined treatment with lucanthone and TRAIL-induced apoptosis. Moreover, lucanthone increased expression level of DR5 mRNA via downregulation of miR-216a-5p. Transfection of miR-216a-5p mimics suppressed the lucanthone-induced DR5 upregulation. Taken together, these results provide the first evidence that lucanthone enhances TRAIL-induced apoptosis through DR5 upregulation by downregulation of miR-216a-5p and DUB3-dependent Mcl-1 downregulation in human renal carcinoma cells.

COMPARE Analysis, a Bioinformatic Approach to Accelerate Drug Repurposing against Covid-19 and Other Emerging Epidemics.

A novel bioinformatic approach for drug repurposing against emerging viral epidemics like Covid-19 is described. It exploits the COMPARE algorithm, a public program from the National Cancer Institute (NCI) to sort drugs according to their patterns of growth inhibitory profiles from a diverse panel of human cancer cell lines. The data repository of the NCI includes the growth inhibitory patterns of more than 55,000 molecules. When candidate drug molecules with ostensible anti-SARS-CoV-2 activities were used as seeds (e.g., hydroxychloroquine, ritonavir, and dexamethasone) in COMPARE, the analysis uncovered several molecules with fingerprints similar to the seeded drugs. Interestingly, despite the fact that the uncovered drugs were from various pharmacological classes (antiarrhythmic, nucleosides, antipsychotic, alkaloids, antibiotics, and vitamins), they were all reportedly known from published literature to exert antiviral activities via different modes, confirming that COMPARE analysis is efficient for predicting antiviral activities of drugs from various pharmacological classes. Noticeably, several of the uncovered drugs can be readily tested, like didanosine, methotrexate, vitamin A, nicotinamide, valproic acid, uridine, and flucloxacillin. Unlike pure in silico methods, this approach is biologically more relevant and able to pharmacologically correlate compounds regardless of their chemical structures. This is an untapped resource, reliable and readily exploitable for drug repurposing against current and future viral outbreaks.

Computational Study of Anticancer Drug Resistance Caused by 10 Topisomerase I Mutations, Including 7 Camptothecin Analogs and Lucanthone.

Although Camptothecin and its analogs as Topoisomerase I poisons can effectively treat cancers, serious drug resistance has been identified for this class of drugs. Recent computational studies have indicated that the mutations near the active binding site of the drug can significantly weaken the drug binding and cause drug resistance. However, only Topotecan and three mutations have been previously analyzed. Here we present a comprehensive binding study of 10 Topoisomerase I mutants (N722S, N722A, D533G, D533N, G503S, G717V, T729A, F361S, G363C, and R364H) and 8 poisons including 7 Camptothecin analogs as well as a new generation Topoisomerase I drug, Lucanthone. Utilizing Glide docking followed by MMGBSA calculations, we determined the binding energy for each complex. We examine the relative binding energy changes with reference to the wild type, which are linked to the degree of drug resistance. On this set of mutant complexes, Topotecan and Camptothecin showed much smaller binding energies than a set of new Camptothecin derivatives (Lurtotecan, SN38, Gimatecan, Exatecan, and Belotecan) currently under clinical trials. We observed that Lucanthone exhibited comparable results to Topotecan and Camptothecin, indicating that it may serve as a promising candidate for future studies as a Topoisomerase I poison. Our docked results on Topotecan were also validated by a set of molecular dynamics simulations. In addition to a good agreement on the MMGBSA binding energy change, our simulation data also shows there is larger conformation fluctuation upon the mutations. These results may be utilized to further advancements of Topoisomerase I drugs that are resistant to mutations.

Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis.

Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53(+/+) and p53(-/-) HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models.

Radiation resistance in glioma cells determined by DNA damage repair activity of Ape1/Ref-1.

Since radiation therapy remains a primary treatment modality for gliomas, the radioresistance of glioma cells and targets to modify their radiation tolerance are of significant interest. Human apurinic endonuclease 1 (Ape1, Ref-1, APEX, HAP1, AP endo) is a multifunctional protein involved in base excision repair of DNA and a redox-dependent transcriptional co-activator. This study investigated whether there is a direct relationship between Ape1 and radioresistance in glioma cells, employing the human U87 and U251 cell lines. U87 is intrinsically more radioresistant than U251, which is partly attributable to more cycling U251 cells found in G2/M, the most radiosensitive cell stage, while more U87 cells are found in S and G1, the more radioresistant cell stages. But observed radioresistance is also related to Ape1 activity. U87 has higher levels of Ape1 than does U251, as assessed by Western blot and enzyme activity assays (approximately 1.5-2 fold higher in cycling cells, and approximately 10 fold higher at G2/M). A direct relationship was seen in cells transfected with CMV-Ape1 constructs; there was a dose-dependent relationship between increasing Ape1 overexpression and increasing radioresistance. Conversely, knock down by siRNA or by pharmacological down regulation of Ape1 resulted in decreased radioresistance. The inhibitors lucanthone and CRT004876 were employed, the former a thioxanthene previously under clinical evaluation as a radiosensitizer for brain tumors and the latter a more specific Ape1 inhibitor. These data suggest that Ape1 may be a useful target for modifying radiation tolerance.

Clonogenicity of human leukemic cells protected from cell-lethal agents by heat shock protein 70.

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.

Inhibition of the human apurinic/apyrimidinic endonuclease (APE1) repair activity and sensitization of breast cancer cells to DNA alkylating agents with lucanthone.

Cells repair DNA damage via four main mechanisms, however, damage induced by alkylators and oxidative damage is predominantly repaired by the DNA base excision repair (BER) pathway. The AP endonuclease, APE1, is one of the main enzymes in the BER pathway. It is abundant in human cells and accounts for nearly all of the abasic site cleavage activity observed in cellular extracts. APE1 expression is elevated in a variety of cancers and a high APE1 expression has been associated with poor outcome to chemoradiotherapy. The small molecule lucanthone has been shown to enhance the killing ability of ionizing radiation in cells and preliminary evidence suggests that lucanthone may inhibit AP endonuclease. Given the role APE1 plays in repairing oxidative and ionizing radiation DNA damage, the reports of lucanthone as an ionizing radiation enhancer and the potential use of lucanthone as an AP endonuclease inhibitor, we examined whether lucanthone could inhibit APE1 endonuclease activity. We report that lucanthone inhibits the repair activity of APE1, but not its redox function or exonuclease activity on mismatched nucleotides. Lucanthone also appears to inhibit exonuclease III family members (APE1 and ExoIII), but not endonuclease IV AP endonucleases, nor bifunctional glycosylase/lyases such as endonuclease VIII or formamidopyrimidine-DNA glycosylase (Fpg). Furthermore, the addition of lucanthone inhibits APE1 repair activity from cellular extracts and enhances the cell killing effect of the laboratory alkylating agent methyl methanesulfonate (MMS) and the clinically relevant agent temozolomide (TMZ). Given these initial findings, it would be of interest to further develop lucanthone as an APE1 inhibitor through the use of structure-function studies as a means of enhancing the sensitization of tumors to chemotherapeutic agents.

Abasic sites in DNA of HeLa cells induced by lucanthone.

Abasic sites in HeLa cell DNA were increased in frequency by exposing the cells to lucanthone. Cell growth in the presence of lucanthone caused progressive accumulation of abasic sites and loss of cellular DNA. After 2 hr in 8 microM lucanthone, the abundance of abasic sites was 2.4 fold greater than the background of 9.9 +/- 2.0 SE abasic sites/10(6) nucleotides; 80 microM lucanthone in the growth medium increased the level 12.6 +/- 2.5 SE fold and decreased the DNA content in HeLa cells to one-half of the value obtained in untreated cells. The frequency of abasic sites in cellular DNA was determined by the aldehyde reactive probe method, with reference to abasic sites created in plasmid pBR322. The ability of lucanthone to inhibit the normal repair of abasic sites might reflect inhibition of apurinic/apyrimidinic endonuclease (HAP1) by the drug, thereby preventing an early step in the base excision repair pathway. Unrepaired abasic sites prevalent after ionizing radiation are cytotoxic lesions that promote DNA strand breaks. These results suggest a rationale for the joint lethal effects of lucanthone and ionizing radiation in cells and the accelerated tumor regression observed in cancer patients who received the combined therapy.

Stimulation of topoisomerase II-mediated DNA cleavage by an indazole analogue of lucanthone.

Lucanthone is an antitumour drug used as an adjuvant in radiation therapy. The drug intercalates into DNA and inhibits topoisomerase II. An indazole analogue of lucanthone (IA-5) was examined for its ability to modulate topoisomerase II-DNA cleavable complex formation in vitro. The drug contains a methylbenzothiopyranoindazole chromophore instead of the methyl-thioxanthenone nucleus of lucanthone. Using a radiolabelled linear plasmid DNA as a substrate, both lucanthone and the indazole analogue were shown to promote the cleavage of DNA by human topoisomerase II. Sequencing experiments with different restriction fragments indicated that the indazole drug promoted DNA cleavage primarily at sites having a C on the 3' side of the cleaved bond (-1 position). By contrast, in the same sequencing methodology lucanthone exerted a much weaker effect on topoisomerase II. The sequence selectivity of IA-5 is reminiscent of that of the anticancer drug mitoxantrone and its anthrapyrazole analogue losoxantrone, which is structurally close to IA-5. Binding to DNA and topoisomerase II inhibition are two distinct processes contributing separately to the cytotoxic activity of the indazole drug.

Topoisomerase inhibition by lucanthone, an adjuvant in radiation therapy.

PURPOSE: To determine whether lucanthone can inhibit human topoisomerases in vitro. METHODS AND MATERIALS: Lucanthone was incubated with human topoisomerases II and I together with their plasmid substrates, to determine if lucanthone interfered with the catalytic activities of topoisomerases and if it enhanced the formation of DNA strand breaks, as determined by agarose gel electrophoresis of the resultant plasmid forms. RESULTS: Incubation of the enzymes with lucanthone inhibited the catalytic activity of topoisomerases II and I. With topoisomerase II, it increased the abundance of DNA double strand breaks (cleavable complexes). CONCLUSION: Lucanthone, like actinomycin D, inhibited topoisomerases II and I. It may act to enhance the yield of DNA double strand breaks in cells through a mechanism of topoisomerase II inhibition.

Studies on carbohydrates extracted from native and chemically treated Biomphalaria alexandrina snails.

1. Carbohydrates were extracted from total tissue extracts of Biomphalaria alexandrina snails and were analyzed to their monosaccharides using GLC. 2. The snails were chemically treated with thioxanthone derivatives (compounds I, II, III) and the change in the monosaccharide constituents of their carbohydrates was investigated. 3. The isolated monosaccharides from native and chemically pretreated snails were injected into mice and their protective effects were examined after infection of mice with cercariae of Schistosoma mansoni. 4. The results showed that the main monosaccharides in carbohydrates of snails were galactose, glucose, fucose and mannose and that chemical treatment caused a drop in the galactose content. 5. Moreover, monosaccharide fractions from snails treated with compound III were the most effective in inducing protection against Schistosoma infection in mice.

Lack of correlation between schistosomicidal and anticholinergic properties of hycanthone and related drugs.

Visual observation of the motor activity of Schistosoma mansoni kept in vitro showed an increase of activity in the presence of hycanthone (HC). In addition, HC caused a delay in the paralytic effects of carbachol. Similar results were observed in the presence of oxamniquine (OXA). The same pattern of motor activity, however, was shown by HC-resistant worms, by Schistosoma japonicum, and by worms exposed to drug precursors (lucanthone and UK-3883), which are not schistosomicidal in vitro. Other analogs with in vitro killing activity (IA-4 and IA-4 N-oxide) showed minimal anticholinergic effects. The anticholinergic effects of HC and OXA were quickly reversible in vitro and in vivo, whereas their antischistosomal effects are irreversible and delayed. Incubation of schistosomes with high concentrations of carbachol or with anticholinergic drugs failed to compete with the schistosomicidal effects of HC. These results are viewed as contradictory to the hypothesis that HC kills schistosomes by blocking their acetylcholine receptors.

Studies on the mode of action of oxamniquine and related schistosomicidal drugs.

Adult Schistosoma mansoni were incubated for 1 hour in vitro with various drugs and then returned into the mesenteric veins of permissive animal hosts. Survival of schistosomes was assessed 3-4 weeks later by portal perfusion. Under these conditions, oxamniquine and hycanthone proved effective in killing S. mansoni, whereas UK-3883, lucanthone and lucanthone-4-desmethyl had no lethal activity. The same drugs which were schistosomicidal in vitro also persistently inhibited DNA, RNA, and protein synthesis in S. mansoni, whereas they were only transiently inhibitory against Schistosoma japonicum, against hycanthone-resistant S. mansoni and against immature worms. When drugs were administered in vivo to infected mice and the synthesis of macromolecules was assayed in vitro on worms obtained 1 or 3 days after treatment, not only oxamniquine and hycanthone, but also UK-3883 and lucanthone, proved effective in inhibiting the synthesis of macromolecules in sensitive--but not in resistant--S. mansoni. It is suggested that oxamniquine, like hycanthone, may exert its schistosomicidal activity by inhibiting nucleic acid synthesis in the parasite.

The effect of lucanthone on sublethal radiation damage, in vivo.

The capacity of the Chinese hamster jejunal crypt cell to accumulate and repair sublethal radiation damage was determined by analyzing the return of the shoulder of the radiation dose-crypt microcolony survival curve (Dr) after a priming dose of 1250 rad. The control split dose crypt cell survival curve exhibited a D0, Dr and "n" of 179 +/- 3 rad, 261 +/- 3 rad and 4.3 respectively; repair of sublethal radiation damage was completed by two hours post-irradiation. The effect of lucanthone (an antischistosomal DNA intercalating agent) on the crypt cell's capacity to accumulate and repair sublethal radiation damage was determined by injecting the drug (100 mg/kg, i.p.) at intervals before irradiation with a priming dose of 1250 rad, followed two hours later by graded doses. Injection coincident with the priming dose of radiation resulted in a 22 rad reduction of the Dr (compared to control Dr). Injection eight hours before the priming dose almost completely inhibited the accumulation and repair of sublethal radiation damage so that the resultant Dr two hours later was only 29 rad (a 232 rad reduction). At no time was the D0 of the crypt cell survival curve affected by lucanthone. These data confirm previous results from whole crypt analysis and LD50/7 analysis that non-toxic concentrations of lucanthone reversibly inhibit the accumulation and repair of sublethal radiation damage in a time-dependent manner with complete inhibition approximately eight hours post-injection. This drug is useful for the study of sublethal radiation damage in vivo and may be beneficial in radiation therapy of cancer when it is desirable to inhibit the repair of sublethal radiation damage.

Lucanthone modification of cyclophosphamide toxicity in the Chinese hamster.

The interaction of lucanthone and cyclophosphamide (CYC) was investigated in the Chinese hamster in terms of the LD50/7 and LD50/30. These values may be indicative of gastrointestinal stem cell depletion and bone marrow stem cell depletion, respectively. When a nonlethal dose of 100 mg/kg lucanthone preceded CYC injection, the LD50/7 for CYC reached its minimum value of 470 mg/kg at a treatment interval of 10 hours. Lucanthone administered simultaneously with CYC had no effect on the control LD50/7 of 750 mg/kg, and by 48 hours after lucanthone administration the LD50/7 had returned to the control value. When CYC administration preceded that of lucanthone, the LD50/7 reached a minimum of value of 610 mg/kg at an interval of 5 hours; however, for the entire sequence it was approximately 640 mg/kg over all intervals up to 48 hours. The LD50/30 for CYC was only slightly reduced by the presence of lucanthone, indicating that bone marrow sensitivity to CYC was only marginally affected by lucanthone. These data indicate that lucanthone may interact with CYC damage in much the same way as it interacts with radiation damage, viz, by reducing cellular capacity to accumulate and repair sublethal damage.

Ring-hydroxylated analogues of lucanthone as antitumor agents.

A series of ring-alkoxylated and ring-hydroxylated analogues of lucanthone was prepared and tested for antitumor activity. The most biologically interesting members of this group were the 7-hydroxylucanthone derivatives, 50 and 51, which gave T/C values in the NCI P-388 antitumor screen of 188 and 265, respectively. The apparent association constants and delta Tm values for a number of analogue-DNA complexes were determined to ascertain whether there was any quantitative correlation with biological activity. The most that can be said is that intercalation may be a necessary but far from sufficient condition for antitumor activity.