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Clinical efficacy of oxaliplatin is frequently limited by severe adverse effects and therapy resistance. Acquired insensitivity to oxaliplatin is, at least in part, associated with elevated levels of glutathione (GSH). In this study we report on an oxaliplatin-based platinum(IV) prodrug, which releases L -buthionine- S , R -sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase, the rate-limiting enzyme in GSH biosynthesis. Two complexes bearing either acetate ( BSO-OxOAc ) or an albumin-binding maleimide ( BSO-OxMal ) as second axial ligand were synthesized and characterized. The in vitro anticancer activity of BSO-OxOAc was massively reduced in comparison to oxaliplatin, proving its prodrug nature. Nevertheless, the markedly lower intracellular oxaliplatin uptake in resistant HCT116/OxR cells was widely overcome by BSO-OxOAc resulting in distinctly reduced resistance levels. Platinum accumulation in organs of a colorectal cancer mouse model revealed higher tumor selectivity of BSO-OxMal as compared to oxaliplatin. This corresponded with increased antitumor activity, resulting in significantly enhanced overall survival. BSO-OxMal -treated tumors exhibited reduced GSH levels, proliferative activity and enhanced DNA damage (pH2AX) compared to oxaliplatin. Conversely, pH2AX staining especially in kidney cells was distinctly increased by oxaliplatin but not by BSO-OxMal . Taken together, our data provide compelling evidence for enhanced tumor specificity of the oxaliplatin(IV)/BSO prodrug. Platinum(IV) complexes have the potential to overcome several limitations and reduce adverse effects of platinum-based cancer therapy. Resistance to clinically approved platinum(II) drugs is, at least in part, associated with elevated levels of glutathione. Here, the authors report on an oxaliplatin-based platinum(IV) prodrug, which releases L -buthionine- S , R -sulfoximine, an inhibitor of the rate-limiting enzyme in glutathione biosynthesis, to circumvent glutathione-based resistance mechanisms.

The tumor suppressor p16INK4a, encoded by CDKN2A, is frequently inactivated in cancer through genetic or epigenetic mechanisms. While promoter hypermethylation is the most common epigenetic cause, aberrant methylation of CDKN2A exon 2 has also been associated with various tumor types. However, analyzing DNA methylation of exon 2 is challenging due to its high sequence similarity with CDKN2B. We developed a pyrosequencing assay to analyze CpGs in CDKN2A exon 2, which was previously found to be hypermethylated in breast cancer. Our novel primer set enabled co-amplification of the homologous regions in CDKN2A, including CpGs 1–24, and CDKN2B CpGs 1–23. By quantifying the proportion of CDKN2A, we could accurately determine methylation levels for CpGs in CDKN2A exon 2. This method was applied to patient-derived glioma cells and commercial breast cancer cell lines. To reveal the role of exon 2 methylation in gene regulation, we additionally examined CDKN2AINK4a promoter methylation and expression at both mRNA and protein levels in breast cancer cell lines. We observed a range of (epi)genetic alterations, including homozygous deletions, transcript-specific expression, and exon 2 skipping. Our findings indicate that both promoter and exon 2 methylation contribute to regulation of CDKN2A expression. This novel method provides a valuable tool for future studies seeking a deeper understanding of CDKN2A regulation in cancer.

Background Gene amplification of MET , which encodes for the receptor tyrosine kinase c-MET, occurs in a variety of human cancers. High c-MET levels often correlate with poor cancer prognosis. Interleukin-like EMT inducer (ILEI) is also overexpressed in many cancers and is associated with metastasis and poor survival. The gene for ILEI, FAM3C , is located close to MET on chromosome 7q31 in an amplification “hotspot”, but it is unclear whether FAMC3 amplification contributes to elevated ILEI expression in cancer. In this study we have investigated FAMC3 copy number gain in different cancers and its potential connection to MET amplifications. Methods FAMC3 and MET copy numbers were investigated in various cancer samples and 200 cancer cell lines. Copy numbers of the two genes were correlated with mRNA levels, with relapse-free survival in lung cancer patient samples as well as with clinicopathological parameters in primary samples from 49 advanced stage colorectal cancer patients. ILEI knock-down and c-MET inhibition effects on proliferation and invasiveness of five cancer cell lines and growth of xenograft tumors in mice were then investigated. Results FAMC3 was amplified in strict association with MET amplification in several human cancers and cancer cell lines. Increased FAM3C and MET copy numbers were tightly linked and correlated with increased gene expression and poor survival in human lung cancer and with extramural invasion in colorectal carcinoma. Stable ILEI shRNA knock-down did not influence proliferation or sensitivity towards c-MET-inhibitor induced proliferation arrest in cancer cells, but impaired both c-MET-independent and -dependent cancer cell invasion. c-MET inhibition reduced ILEI secretion, and shRNA mediated ILEI knock-down prevented c-MET-signaling induced elevated expression and secretion of matrix metalloproteinase (MMP)-2 and MMP-9. Combination of ILEI knock-down and c-MET-inhibition significantly reduced the invasive outgrowth of NCI-H441 and NCI-H1993 lung tumor xenografts by inhibiting proliferation, MMP expression and E-cadherin membrane localization. Conclusions These novel findings suggest MET amplifications are often in reality MET-FAM3C co-amplifications with tight functional cooperation. Therefore, the clinical relevance of this frequent cancer amplification hotspot, so far dedicated purely to c-MET function, should be re-evaluated to include ILEI as a target in the therapy of c-MET-amplified human carcinomas.

Due to their high biological activity, thiosemicarbazones have been developed for treatment of diverse diseases, including cancer, resulting in multiple clinical trials especially of the lead compound Triapine. During the last years, a novel subclass of anticancer thiosemicarbazones has attracted substantial interest based on their enhanced cytotoxic activity. Increasing evidence suggests that the double-dimethylated Triapine derivative Me 2 NNMe 2 differs from Triapine not only in its efficacy but also in its mode of action. Here we show that Me 2 NNMe 2 - (but not Triapine)-treated cancer cells exhibit all hallmarks of paraptotic cell death including, besides the appearance of endoplasmic reticulum (ER)-derived vesicles, also mitochondrial swelling and caspase-independent cell death via the MAPK signaling pathway. Subsequently, we uncover that the copper complex of Me 2 NNMe 2 (a supposed intracellular metabolite) inhibits the ER-resident protein disulfide isomerase, resulting in a specific form of ER stress based on disruption of the Ca 2+ and ER thiol redox homeostasis. Our findings indicate that compounds like Me 2 NNMe 2 are of interest especially for the treatment of apoptosis-resistant cancer and provide new insights into mechanisms underlying drug-induced paraptosis.

Tyrosine kinase inhibitors revolutionized cancer therapy but still evoke strong adverse effects that can dramatically reduce patients’ quality of life. One possibility to enhance drug safety is the exploitation of prodrug strategies to selectively activate a drug inside the tumor tissue. In this study, we designed a prodrug strategy for the approved c-MET, ALK, and ROS1 tyrosine kinase inhibitor crizotinib. Therefore, a boronic-acid trigger moiety was attached to the 2-aminopyridine group of crizotinib, which is a crucial position for target kinase binding. The influence of the modifications on the c-MET- and ALK-binding ability was investigated by docking studies, and the strongly reduced interactions could be confirmed by cell-free kinase inhibition assay. Furthermore, the newly synthesized compounds were tested for their activation behavior with H2O2 and their stability in cell culture medium and serum. Finally, the biological activity of the prodrugs was investigated in three cancer cell lines and revealed a good correlation between activity and intrinsic H2O2 levels of the cells for prodrug A. Furthermore, the activity of this prodrug was distinctly reduced in a non-malignant, c-MET expressing human lung fibroblast (HLF) cell line.

Following two orthogonal synthetic routes, a series of all four possible A-ring amino derivatives of the natural product Luotonin A (a known Topoisomerase I inhibitor) was synthesized. In both strategies, intramolecular cycloaddition reactions are the key step. The target compounds were obtained in good yields by mild catalytic transfer hydrogenation of the corresponding nitro precursors. In-vitro evaluation of the antiproliferative activity towards human tumor cell lines revealed the 4-amino compound (5b) to be the most effective agent, showing an interesting profile of cytotoxic activity. Among other effects, a significant G2/M cell cycle arrest was observed for this compound, suggesting that either Topoisomerase I is not the only biological target, or that some atypical mechanism is responsible for inhibition of this enzyme.

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG). In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity.

Clinical failure of novel drugs is often related to their rapid metabolism and excretion. This highlights the importance of elucidation of their pharmacokinetic profile already at the preclinical stage of drug development. Triapine, the most prominent representative of α-N-heterocyclic thiosemicarbazones, was investigated in more than 30 clinical phase I/II trials, but the results against solid tumors were disappointing. Recent investigations from our group suggested that this is, at least partially, based on the fast metabolism and excretion. In order to establish more detailed structure/activity/metabolism relationships, herein a panel of 10 different Triapine derivatives was investigated for their metabolic pathways. From the biological point of view, the panel consists of terminally dimethylated thiosemicarbazones with nanomolar IC50 values, derivatives with micromolar cytotoxicities comparable to Triapine and a completely inactive representative. To study the oxidative metabolism, a purely instrumental approach based on electrochemistry/mass spectrometry was applied and the results were compared to the data obtained from microsomal incubations. Overall, the investigated thiosemicarbazones underwent the phase I metabolic reactions dehydrogenation, hydroxylation, oxidative desulfuration (to semicarbazone and amidrazone) and demethylation. Notably, dehydrogenation resulted in a ring-closure reaction with formation of thiadiazoles. Although strong differences between the metabolic pathways of the different thiosemicarbazones were observed, they could not be directly correlated to their cytotoxicities. Finally, the metabolic pathways for the most cytotoxic compound were elucidated also in tissues collected from drug-treated mice, confirming the data obtained by electrochemical oxidation and microsomes. In addition, the in vivo experiments revealed a very fast metabolism and excretion of the compound.Graphical abstractStructure/activity/metabolisation relationships for 10 anticancer thiosemicarbazones were established using electrochemical oxidation coupled to mass spectrometry (EC-MS) and human liver microsomes analyzed by LC-MS

The tree fern Metaxya rostrata (Kunth) C. Presl is common in the rainforests of Central and South America, where suspensions of the dried rhizome are traditionally used to treat intestinal diseases. Two compounds from this plant, 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB), have been shown to be biologically highly active against colorectal cancer (CRC) cells in previous studies. The current investigation resulted in the isolation of the previously undescribed methylated xanthones 2-deprenyl-6-O-methyl-7-hydroxy-rheediaxanthone B, 2-deprenyl-5-O-methyl-7-methoxy-rheediaxanthone B, 2-deprenyl-5-O-methyl- 7-hydroxy-rheediaxanthone B and 2-deprenyl-7-methoxy-rheediaxanthone B. All compounds were isolated by column chromatography, structures were elucidated by one- and two-dimensional NMR-experiments and the identities of the compounds were confirmed by LC-HRMS. In logarithmically growing SW480 CRC cell cultures, cytotoxicity by neutral red uptake and MTT assays as well as caspase activation was analyzed. Cellular targets were examined by Western blot, and topoisomerase I (topo I) inhibition potential was tested. Comparing the structure-activity relationship with XB and OH-XB, the monomethylated derivatives showed qualitatively similar effects/mechanisms to their nonmethylated analogues, while dimethylation almost abolished the activity. Inhibition of topo I was dependent on the presence of an unmethylated 7-OH group.

Background Osteosarcoma is the most frequent primary malignant bone tumor. Although survival has distinctly increased due to neoadjuvant chemotherapy in the past, patients with metastatic disease and poor response to chemotherapy still have an adverse prognosis. Hence, development of new therapeutic strategies is still of utmost importance. Methods Anticancer activity of KP46 against osteosarcoma cell models was evaluated as single agent and in combination approaches with chemotherapeutics and Bcl-2 inhibitors using MTT assay. Underlying mechanisms were tested by cell cycle, apoptosis and autophagy assays. Results KP46 exerted exceptional anticancer activity at the nanomolar to low micromolar range, depending on the assay format, against all osteosarcoma cell models with minor but significant differences in IC 50 values. KP46 treatment of osteosarcoma cells caused rapid loss of cell adhesion, weak cell cycle accumulation in S-phase and later signs of apoptotic cell death. Furthermore, already at sub-cytotoxic concentrations KP46 reduced the migratory potential of osteosarcoma cells and exerted synergistic effects with cisplatin, a standard osteosarcoma chemotherapeutic. Moreover, the gallium compound induced signs of autophagy in osteosarcoma cells. Accordingly, blockade of autophagy by chloroquine but also by the Bcl-2 inhibitor obatoclax increased the cytotoxic activity of KP46 treatment significantly, suggesting autophagy induction as a protective mechanism against KP46. Conclusion Together, our results identify KP46 as a new promising agent to supplement standard chemotherapy and possible future targeted therapy in osteosarcoma.