Former studies already revealed the anti-neoplastic properties of the anti-infective agent Taurolidine (TRD) against many tumor species in vitro and in vivo. Its anti-proliferative and cell death inducing capacity is largely due to its main derivative Taurultam (TRLT). In this study it could be demonstrated, that substance 2250 - a newly defined innovative structural analogue of TRLT - exhibits an anti-neoplastic effect on malignant pancreatic carcinoma in vitro and in vivo. The anti-neoplastic potential of substance 2250 as well as its mode of action was demonstrated in extensive in vitro analysis, followed by successful and effective in vivo testings, using xenograft models derived from established pancreatic cancer cell lines as well as patient derived tissue. Our functional analysis regarding the role of oxidative stress (ROS) and caspase activated apoptosis showed, that ROS driven programmed cell death (PCD) is the major mechanisms induced by substance 2250 in pancreatic carcinoma. What is strongly relevant towards clinical practice is especially the observed inhibition of patient derived pancreatic cancer tumor growth in mice treated with this new substance in combination with its sharply higher metabolic stability. These encouraging results provide new therapeutical opportunities in pancreatic cancer treatment and build the basis for further functional analysis as well as first clinical studies for this promising agent.

GP‐2250, a novel anticancer agent, severely limits the energy metabolism, as demonstrated by the inhibition of hexokinase 2 and glyceraldehyde‐3‐phosphate dehydrogenase and a decrease of ATP. Rescue experiments with supplementary pyruvate or oxaloacetate demonstrated that a TCA cycle deficit largely contributed to cytotoxicity. Activation of the energy‐deficit sensor, AMP‐dependent protein kinase, was associated with increased phosphorylation of acetyl‐CoA carboxylase and Raptor, pointing to a possible deficit in the synthesis of fatty acids and proteins as essential cell components. Binding of p65 to DNA was dose‐dependently reduced in nuclear lysates. A transcriptional deficit of NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) was substantiated by the downregulation of cyclin D1 and of the anti‐apoptotic Bcl2, in line with reduction in tumour cell proliferation and induction of apoptosis, respectively. The upregulation of p53 concomitant with an excess of ROS supported apoptosis. Thus, the anticancer activity of GP‐2250 is a result of disruption of energy metabolism and inhibition of tumour promotion by NF‐κB.

Neuroendocrine carcinoma of the pancreas (pNEC) is an aggressive form of neuroendocrine tumor characterized by a rising incidence without an increase in survival rates. GP-2250 is an oxathiazinane derivate possessing antineoplastic effects, especially in combination with Gemcitabine on the pancreatic adenocarcinoma. The cytotoxic effects of the monotherapy of GP-2250 (GP-2250_mono) and Gemcitabine (Gem_mono), as well as the combination therapy of both, were studied in vitro using an MTT-assay on the QGP-1 and BON-1 cell lines, along with in vivo studies on a murine xenograft model of QGP-1 and a patient-derived xenograft model (PDX) of Bo99. In vitro, Gem_mono and GP-2250_mono showed a dose-dependent cytotoxicity. The combination of GP-2250 and Gemcitabine exhibited highly synergistic effects. In vivo, the combination therapy obtained a partial response in QGP-1, while GP-2250_mono and Gem_mono showed progressive disease or stable disease, respectively. In Bo99 PDX, the combination therapy led to a partial response, while the monotherapy resulted in progressive disease. No development of secondary resistances was observed, as opposed to monotherapy. This study was the first to evaluate the effects of the emerging substance GP-2250 on pNEC. The substance showed synergism in combination with Gemcitabine. The combination therapy proved to be effective in vitro and in vivo, without the development of secondary resistances.

Background: Even in the novel immunotherapy era, Merkel cell carcinoma (MCC) remains challenging in its treatment. Apart from Merkel cell polyomavirus (MCPyV) associated MCC, this cancer is linked in about 20% of cases to ultraviolet-induced mutational burden frequently causing aberrations in Notch and PI3K/AKT/mTOR signalling pathways. The recently developed agent GP-2250 is capable to inhibit growth of cells of different cancers, including pancreatic neuroendocrine tumors. The objective of the present study was to investigate the effects of GP-2250 on MCPyV-negative MCC cells. Methods: Methods We employed three cell lines (MCC13, MCC14.2, MCC26) which were exposed to different GP-2250doses. GP-2250’s effects on cell viability, proliferation, and migration were evaluated by means of MTT, BrdU, and scratch assays, respectively. Flow cytometry was performed for the evaluation of apoptosis and necrosis. Western blotting was implemented for the determination of AKT, mTOR, STAT3, and Notch1 protein expression. Results: Cell viability, proliferation, and migration decreased with increasing GP-2250 doses. Flow cytometry revealed a dose response to GP-2250 in all three MCC cell lines. While the viable fraction decreased, the share of necrotic and in a smaller amount the apoptotic cells increased. Regarding Notch1, AKT, mTOR, and STAT3 expression a comparatively time- and dose-dependent decrease of protein expression in the MCC13 and MCC26 cell lines was observed. By contrast, Notch1, AKT, mTOR, and STAT3 expression in MCC14.2 was scarcely altered or even increased by the three dosages of GP-2250 applied. Conclusions: The present study indicates GP-2250 having anti-neoplastic effects in MCPyV-negative tumor cells in regard to viability, proliferation, and migration. Moreover, the substance is capable of downregulating protein expression of aberrant tumorigenic pathways in MCPyV-negative MCC cells.

Purpose: The Oxathiazinane substance class is characterized by a high diversity of chemical structures yet to be fully investigated. Our research group recently proved that the 1.4.5-oxathiazine-4.4-dioxide, known as substance GP-2250, possesses antineoplastic properties as shown on pancreatic carcinoma. This current study aims to gain insights into the structure and activity relationship of a series of different Oxathiazinanes regarding their antineoplastic activity and the potential correlation with antibacterial activity. We investigated the newly synthesized Oxathiazinane derivatives: 2255, 2256, 2287, 2289, 2293 and 2296 in comparison to GP-2250. Methods: The antineoplastic effect was evaluated in different cancer entities (breast, skin, pancreas and colon cancer cell lines) by viability, proliferation, and cell migration assays in vitro. Disc diffusion tests were performed on various bacteria strains to examine the antibacterial potential. Additionally, reactive oxygen species (ROS) assays were conducted to investigate mechanistic aspects. Results: The substances GP-2250, 2293, 2289 and 2296 not only showed antineoplastic activity in four different cancer entities but also antibacterial effects, as tested on multiple bacteria strains including MRSA (Methicillin-resistant Staphylococcus aureus). Furthermore, these substances also induced high ROS levels up to 110% in the treated cancer cell lines compared to untreated control cells. These results indicate a correlation between an antineoplastic capacity and antibacterial properties of these derivatives. Both activities appear to be ROS driven. The Oxathiazinane derivatives 2255, 2256 and 2287 lacked both, antineoplastic and antibacterial activity. Conclusion: Thus, a comparable structure activity relationship became apparent for both the antineoplastic and antibacterial activity.

Enhanced glycolysis (Warburg effect) driven by the BRAF oncogene, dysregulated GAPDH expression, and activation of the PI3K/AKT/mTOR signaling pathway may significantly contribute to the resistance-targeted therapy of BRAF-mutated melanomas. Therefore, we aimed to study for the first time the anti-tumor activity of the GAPDH inhibitor GP-2250 in BRAF-mutated melanoma cell lines and benign melanocytes. We employed three melanoma cell lines and one primary melanocyte cell line (Ma-Mel-61a, Ma-Mel-86a, SH-4 and ATCC-PCS-200-013, respectively), which were exposed to different GP-2250 doses. GP-2250’s effects on cell proliferation and viability were evaluated by means of the BrdU and MTT assays, respectively. The RealTime-Glo Annexin V Apoptosis and Necrosis Assay was performed for the evaluation of apoptosis and necrosis induction. RT-PCR and western blotting were implemented for the determination of AKT and STAT3 gene and protein expression analyses, respectively. The melanoma cell lines showed a dose-dependent response to GP-2250 during BrDU and MTT testing. The RealTime-Glo Annexin V assay revealed the heterogenous impact of GP-2250 on apoptosis as well as necrosis. With respect to the melanoma cell lines Ma-Mel-86a and SH-4, the responses and dosages were comparable to those used for the MTT viability assay. Using the same dose range of GP-2250 administered to melanoma cells, however, we observed neither the noteworthy apoptosis nor necrosis of GP-2250-treated benign melanocytes. The gene expression profiles in the melanoma cell lines for AKT and STAT3 were heterogenous, whereby AKT as well as STAT3 gene expression were most effectively downregulated using the highest GP-2250 doses. Immunoblotting revealed that there was a time-dependent decrease in protein expression at the highest GP-2250 dose used, whereas a time- as well as dose-dependent AKT decrease was predominantly observed in Ma-Mel-61a. The STAT3 protein expression of Ma-Mel-86a and SH-4 was reduced in a time-dependent pattern at lower and moderate doses. STAT3 expression in Ma-Me-61a was barely altered by GP-2250. In conclusion, GP-2250 has anti-neoplastic effects in BRAF-mutated melanoma cell lines regarding tumor cell viability, proliferation, and apoptosis/necrosis. GP-2250 is able to downregulate the gene and protein expression of aberrant tumorigenic pathways in melanoma cell lines. Since GP-2250 is a GAPDH inhibitor, the substance may be a promising combination therapy for tumors presenting the Warburg effect, such as melanoma.