Preclinical Synergistic Combination Therapy of Lurbinectedin with Irinotecan and 5-Fluorouracil in Pancreatic Cancer.

Pancreatic cancer is a devastating disease with a poor prognosis. Novel chemotherapeutics in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin (PM01183; LY-01017) received FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. Lurbinectedin stalls and degrades RNA Polymerase II and introduces breaks in DNA, causing subsequent apoptosis. We now demonstrate lurbinectedin's highly efficient killing of human-derived pancreatic tumor cell lines PANC-1, BxPC-3, and HPAF-II as a single agent. We further demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in the synergistic killing of pancreatic tumor cells. Western blot analysis of combination therapy indicates an upregulation of γH2AX, a DNA damage marker, and the Chk1/ATR pathway, which is involved in replicative stress and DNA damage response. We further demonstrate that the triple combination between lurbinectedin, irinotecan, and 5-fluorouracil (5-FU) results in a highly efficient killing of tumor cells. Our results are developing insights regarding molecular mechanisms underlying the therapeutic efficacy of a novel combination drug treatment for pancreatic cancer.

Targeting sphingosine kinase 1 in p53KO thymic lymphoma.

Sphingosine kinase 1 (SK1) is a key sphingolipid enzyme that is upregulated in several types of cancer, including lymphoma which is a heterogenous group of malignancies. Treatment for lymphoma has improved significantly by the introduction of new therapies; however, subtypes with tumor protein P53 (p53) mutations or deletion have poor prognosis, making it critical to explore new therapeutic strategies in this context. SK1 has been proposed as a therapeutic target in different types of cancer; however, the effect of targeting SK1 in cancers with p53 deletion has not been evaluated yet. Previous work from our group suggests that loss of SK1 is a key event in mediating the tumor suppressive effect of p53. Employing both genetic and pharmacological approaches to inhibit SK1 function in Trp53KO mice, we show that targeting SK1 decreases tumor growth of established p53KO thymic lymphoma. Inducible deletion of Sphk1 or its pharmacological inhibition drive increased cell death in tumors which is accompanied by selective accumulation of sphingosine levels. These results demonstrate the relevance of SK1 in the growth and maintenance of lymphoma in the absence of p53 function, positioning this enzyme as a potential therapeutic target for the treatment of tumors that lack functional p53.

Promotion of collagen mineralization and dentin repair by succinates.

Biomineralization of collagen fibers is regulated by non-collagenous proteins and small biomolecules, which are essential in bone and teeth formation. In particular, small biomolecules such as succinic acid (SA) exist at a high level in hard tissues, but their role is yet unclear. Here, our work demonstrated that SA could significantly promote intrafibrillar mineralization in two- and three-dimensional collagen models, where the relative mineralization rate was 16 times faster than the control group. Furthermore, the FTIR spectra and isothermal experimental results showed that collagen molecules could interact with SA via a hydrogen bond and that the interaction energy was about 4.35 kJ mol-1. As expected, the SA-pretreated demineralized dentin obtained full remineralization within two days, whereas it took more than four days in the control group, and their mechanical properties were considerably enhanced compared with those of the demineralized one. The possible mechanism of the promotion effect of SA was ultimately illustrated, with SA modification strengthening the capacity of the collagen matrix to attract more calcium ions, which might create a higher local concentration that could accelerate the mineralization of collagen fibers. These findings not only advance the understanding of the vital role of small biomolecules in collagen biomineralization but also facilitate the development of an effective strategy to repair hard tissues.

Preclinical studies with ONC201/TIC10 and lurbinectedin as a novel combination therapy in small cell lung cancer (SCLC).

The American Cancer Society estimates that ~15% of all lung cancers are categorized as small cell lung cancer (SCLC) with an overall five-year survival rate of less than 7%. Due to disease aggressiveness, more other malignancies, the standard of care is based on clinical efficacy rather than helpful biomarkers. Lurbinectedin is a small molecule RNA polymerase II inhibitor that binds the minor groove of DNA to induce double-strand breaks. Lurbinectedin has efficacy towards SCLC cells at sub-nM concentration and received accelerated FDA approval in 2020 for metastatic SCLC that progressed on platinum-based therapy. ONC201/TIC10 is a TRAIL pathway-inducing compound that with demonstrated clinical efficacy in H3K27M-mutated diffuse midline glioma and neuroendocrine tumors, in early phase clinical trials. We hypothesized that combining ONC201 and lurbinectedin may yield synergistic and targeted killing of SCLC cells. SCLC cell lines H1048, H1105, H1882, and H1417 were treated with ONC201 and lurbinectedin and cell viability was determined using a CellTiter-Glo assay using varying drug concentrations. Synergistic growth inhibition of SCLC cells was noted with combination of ONC201 and lurbinectedin. Induction of the integrated stress response mediator ATF4 and CHOP was observed with ONC201 and lurbinectedin along with induction of PARP cleavage indicative of apoptosis in response to cellular stress. Additionally, SCLC lines treated with the combination therapy displayed increased DNA breakage-related proteins such as phosphorylated Chk-1, Wee1 and γ-H2AX. Combination index revealed the most potent synergy occurred at the concentrations of 0.16 µM ONC201 and 0.05 nM lurbinectedin in the H1048 cell line, demonstrating highly efficient and selective killing of these tumor cells in vitro. While these therapies showed potency against the cell lines derived from SCLC patients, it is noteworthy that the combination showed significantly less toxicity to healthy human lung epithelial cells. Future studies could explore the combination of ONC201 and lurbinectedin in SCLC cell lines, SCLC patient-derived organoids, other tumor types, including in vivo studies and clinical translation.

Hydroxypropylmethylcellulose as a film and hydrogel carrier for ACP nanoprecursors to deliver biomimetic mineralization.

Demineralization of hard tooth tissues leads to dental caries, which cause health problems and economic burdens throughout the world. A biomimetic mineralization strategy is expected to reverse early dental caries. Commercially available anti-carious mineralizing products lead to inconclusive clinical results because they cannot continuously replenish the required calcium and phosphate resources. Herein, we prepared a mineralizing film consisting of hydroxypropylmethylcellulose (HPMC) and polyaspartic acid-stabilized amorphous calcium phosphate (PAsp-ACP) nanoparticles. HPMC which contains multiple hydroxyl groups is a film-forming material that can be desiccated to form a dry film. In a moist environment, this film gradually changes into a gel. HPMC was used as the carrier of PAsp-ACP nanoparticles to deliver biomimetic mineralization. Our results indicated that the hydroxyl and methoxyl groups of HPMC could assist the stability of PAsp-ACP nanoparticles and maintain their biomimetic mineralization activity. The results further demonstrated that the bioinspired mineralizing film induced the early mineralization of demineralized dentin after 24 h with increasing mineralization of the whole demineralized dentin (3-4 µm) after 72-96 h. Furthermore, these results were achieved without any cytotoxicity or mucosa irritation. Therefore, this mineralizing film shows promise for use in preventive dentistry due to its efficient mineralization capability.

A Milk-Fat Based Diet Increases Metastasis in the MMTV-PyMT Mouse Model of Breast Cancer.

A high-fat diet (HFD) and obesity are risk factors for many diseases including breast cancer. This is particularly important with close to 40% of the current adult population being overweight or obese. Previous studies have implicated that Mediterranean diets (MDs) partially protect against breast cancer. However, to date, the links between diet and breast cancer progression are not well defined. Therefore, to begin to define and assess this, we used an isocaloric control diet (CD) and two HFDs enriched with either olive oil (OOBD, high in oleate, and unsaturated fatty acid in MDs) or a milk fat-based diet (MFBD, high in palmitate and myristate, saturated fatty acids in Western diets) in a mammary polyomavirus middle T antigen mouse model (MMTV-PyMT) of breast cancer. Our data demonstrate that neither MFBD or OOBD altered the growth of primary tumors in the MMTV-PyMT mice. The examination of lung metastases revealed that OOBD mice exhibited fewer surface nodules and smaller metastases when compared to MFBD and CD mice. These data suggest that different fatty acids found in different sources of HFDs may alter breast cancer metastasis.

Loss of sphingosine kinase 1 increases lung metastases in the MMTV-PyMT mouse model of breast cancer.

Breast cancer is a very heterogeneous disease, and ~30% of breast cancer patients succumb to metastasis, highlighting the need to understand the mechanisms of breast cancer progression in order to identify new molecular targets for treatment. Sphingosine kinase 1 (SK1) has been shown to be upregulated in patients with breast cancer, and several studies have suggested its involvement in breast cancer progression and/or metastasis, mostly based on cell studies. In this work we evaluated the role of SK1 in breast cancer development and metastasis using a transgenic breast cancer model, mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT), that closely resembles the characteristics and evolution of human breast cancer. The results show that SK1 deficiency does not alter tumor latency or growth, but significantly increases the number of metastatic lung nodules and the average metastasis size in the lung of MMTV-PyMT mice. Additionally, analysis of Kaplan-Meier plotter of human disease shows that high SK1 mRNA expression can be associated with a better prognosis for breast cancer patients. These results suggest a metastasis-suppressing function for SK1 in the MMTV-PyMT model of breast cancer, and that its role in regulating human breast cancer progression and metastasis may be dependent on the breast cancer type.

A novel porous silica-zirconia coating for improving bond performance of dental zirconia.

OBJECTIVES: To coat a zirconia surface with silica-zirconia using a dip-coating technique and evaluate its effect on resin-zirconia shear bond strength (SBS). METHODS: A silica-zirconia suspension was prepared and used to coat a zirconia surface using a dip-coating technique. One hundred and eighty-nine zirconia disks were divided into three groups according to their different surface treatments (polishing, sandblasting, and silica-zirconia coating). Scanning electron microscopy (SEM), energy dispersive X-ray (EDX), and X-ray diffraction (XRD) were used to analyze the differently treated zirconia surfaces. Different primer treatments (Monobond N, Z-PRIME Plus, and no primer) were also applied to the zirconia surfaces. Subsequently, 180 composite resin cylinders (Filtek Z350) were cemented onto the zirconia disks with resin cement (RelyX Ultimate). The SBS was measured after water storage for 24 h or 6 months. The data were analyzed by two-way analysis of variance (ANOVA). RESULTS: SEM and EDX showed that the silica-zirconia coating produced a porous layer with additional Si, and XRD showed that only tetragonal zirconia was on the silica-zirconia-coating surface. Compared with the control group, the resin-zirconia SBSs of the sandblasting group and silica-zirconia-coating group were significantly increased (P<0.05). The silica-zirconia coating followed by the application of Monobond N produced the highest SBS (P<0.05). Water aging significantly reduced the resin-zirconia SBS (P<0.05). CONCLUSIONS: Dip-coating with silica-zirconia might be a feasible way to improve resin-zirconia bonding.

Polyelectrolyte-calcium complexes as a pre-precursor induce biomimetic mineralization of collagen.

Polyelectrolytes such as polyaspartic acid (PAsp) are critical in biomimetic mineralization as stabilizers of amorphous calcium phosphate (ACP) precursors and as nucleation inhibitors similar to non-collagenous proteins (NCPs). Nevertheless, the application of polyelectrolyte-calcium complexes as a pre-precursor, such as PAsp-Ca complexes, in the mineralization of collagen is unexplored. Herein, we propose a polyelectrolyte-Ca complex pre-precursor (PCCP) process for collagen mineralization. By combining three-dimensional (3D) STORM, potential measurements, and cryogenic transmission electron microscopy with molecular dynamics simulations, we show that liquid-like electropositive PAsp-Ca complexes along with free calcium ions infiltrate electronegative collagen fibrils. The PAsp-Ca complexes are immobilized within the fibrils via chelation and hydrogen bonds, and outward movement of free calcium ions is prevented while phosphate and hydroxide are recruited through electrostatic attractions. Afterwards, ACP instantly forms and gradually crystallizes. The PCCP process not only unites two distinct crystallization pathways (classical (free Ca/P ions) and non-classical (polyelectrolyte-Ca complexes)), but also provides a novel strategy for rapid biomimetic mineralization of collagen.

Therapeutic Management of Demineralized Dentin Surfaces Using a Mineralizing Adhesive To Seal and Mineralize Dentin, Dentinal Tubules, and Odontoblast Processes.

Dentin hypersensitivity is attributable to the exposed dentin and its patent tubules. We proposed the therapeutic management of demineralized dentin surfaces using a mineralizing adhesive to seal and remineralize dentin, dentinal tubules, and odontoblast processes. An experimental self-etch adhesive and a mineralizing adhesive consisting of the self-etch adhesive and 20 wt % poly-aspartic acid-stabilized amorphous calcium phosphate (PAsp-ACP) nanoparticles were prepared and characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and scanning electron microscopy. After 60 acid-etched midcoronal dentin disks were treated with distilled water (control), a desensitizing agent (Gluma), the experimental self-etch adhesive, and the mineralizing adhesive, dentin permeability was measured and mineralization was evaluated by Raman, FTIR, XRD, TEM, and selected-area electron diffraction, irrespective of abrasive and acidic challenges. In vitro cytotoxicity of the adhesive and the mineralizing adhesive was assessed by Cell Counting Kit-8. The mineralizing adhesive possessed excellent biocompatibility. We proposed a hybrid mineralization layer composed of the light-cured mineralizing adhesive and the mineralized dentin surfaces, as well as interiorly mineralized resin tags and odontoblast processes inside of the dentinal tubules. This hybrid mineralization not only reduced dentin permeability but also resisted abrasive and acidic attacks.

A novel fluorescent adhesive-assisted biomimetic mineralization.

We propose a novel fluorescent adhesive-assisted biomimetic mineralization strategy, based on which 1 wt% of sodium fluorescein and 25 wt% of polyacrylic acid stabilized amorphous calcium phosphate (PAA-ACP) nanoparticles were incorporated into a mild self-etch adhesive (Clearfil S3 Bond) as a fluorescent mineralizing adhesive. The characterization of the PAA-ACP nanoparticles indicates that they were spherical particles clustered together, each particle with a diameter of approximately 20-50 nm, in a metastable phase with two characteristic absorption peaks (1050 cm-1 and 580 cm-1). Our results suggest that the fluorescent mineralizing adhesive was non-cytotoxic with minimal esthetic interference and its fluorescence intensity did not significantly decrease within 6 months. Our data reveal that the fluorescent mineralizing adhesive could induce the extra- and intra-fibrillar remineralization of the reconstituted type I collagen, the demineralized enamel and dentin substrate. Our data demonstrate that a novel fluorescent adhesive-assisted biomimetic mineralization strategy will pave the way to design and produce anti-carious materials for the prevention of dental caries.

Self-Etch Adhesive as a Carrier for ACP Nanoprecursors to Deliver Biomimetic Remineralization.

Lab biomineralization should be carried out in an actual clinical practice. This study evaluated self-etch adhesive as a carrier for amorphous calcium phosphate (ACP) nanoprecursors to continuously deliver biomimetic remineralization of self-assembly type I collagen and demineralized dentin. Si-containing ACP particles (Si-ACP) stabilized with polyaspartic acid (PAsp) were synthesized and characterized by transmission electron microscopy (TEM), scanning electron microscopy-energy-dispersive X-ray spectroscopy, Fourier transform infrared analysis, X-ray powder diffractometry, and X-ray phototelectron spectroscopy. The biomimetic remineralization of single-layer reconstituted type I collagen fibrils and demineralized dentin was analyzed by using two one-bottle self-etch dentin adhesives (Clearfil S3 Bond (S3), Kurraray-Noritake; Adper Easy One (AEO), 3 M ESPE) as a carrier loaded (or not, in the case of the control) with 25 wt % of Si-ACP particles. In vitro cytotoxicity assessed by the Cell Counting Kit-8 indicated that the Si-ACP particles had no adverse effect on cell viability. The capacity for Ca and P ions release from cured Si-ACP-containing adhesives (S3, AEO) was evaluated by inductively coupled plasma-atomic emission spectrometry, revealing the successively increasing release of Ca and P ions for 28 days. The intra- and extrafibrillar remineralization of type I collagen and demineralized dentin was confirmed by TEM and selected-area electron diffraction when the adhesives were used as a carrier loaded with Si-ACP particles. Therefore, we propose self-etch adhesive as a novel carrier for ACP nanoprecursors to continuously deliver biomimetic remineralization.

Susceptibility to ATP depletion of primary proximal tubular cell cultures derived from mice lacking either the α1 or the α2 isoform of the catalytic domain of AMPK.

BACKGROUND: The purpose of this study was to determine whether AMPK influences the survival of primary cultures of mouse proximal tubular (MPT) cells subjected to metabolic stress. Previous studies, using an immortalized MPT cell line, suggest that AMPK is activated during metabolic stress, and ameliorates stress-induced apoptosis of these cells. METHODS: Primary MPT cells were cultured from AMPK knockout (KO) mice lacking either the α1 or the α2 isoform of the catalytic domain of AMPK. MPT cells were subjected to ATP depletion using antimycin A. RESULTS: Surprisingly, there was no difference in the amount of death induced by metabolic stress of MPT cells from either type of AMPK KO mice compared to its WT control. Moreover, inhibition of the activity of the α1 isoform in primary MPT cells from α2-/- mice (pharmacologically, via compound C) or inhibition of the α2 isoform in primary MPT cells from α1-/- mice (molecularly, via knockdown) both decreased cell viability equivalently in response to metabolic stress. The explanation for this unexpected result appears to be an adaptive increase in expression of the non-deleted α-isoform. As a consequence, total α-domain expression (i.e. α1 + α2), is comparable in kidney cortex and in cultured MPT cells derived from either type of KO mouse versus its WT control. Importantly, each α-isoform appears able to compensate fully for the absence of the other, with respect to both the phosphorylation of downstream targets of AMPK and the amelioration of stress-induced cell death. CONCLUSIONS: These findings not only confirm the importance of AMPK as a pro-survival kinase in MPT cells during metabolic stress, but also show, for the first time, that each of the two α-isoforms can substitute for the other in MPT cells from AMPK KO mice with regard to amelioration of stress-induced loss of cell viability.

AMPK protects proximal tubular cells from stress-induced apoptosis by an ATP-independent mechanism: potential role of Akt activation.

We examined the role of AMP-activated protein kinase (AMPK) in modulating the viability of cultured kidney proximal tubular cells subjected to metabolic stress induced by either dextrose deprivation, inhibition of glycolysis, or inhibition of mitochondrial respiration. We used BU.MPT cells, a conditionally immortalized kidney epithelial cell line derived from the proximal tubules of transgenic mice bearing a temperature-sensitive mutation of the simian virus 40 large-tumor antigen. All three forms of metabolic stress increased the phosphorylation and activity of AMPK. Activation of AMPK led to changes in the phosphorylation of two downstream targets of AMPK, acetyl coenzyme A carboxylase and p70 S6 kinase. Inhibition of AMPK, either pharmacologically with compound C (CC) or by gene silencing, significantly increased the amount of apoptosis in response to all three forms of metabolic stress. Although the amount of apoptosis was directly related to the severity of ATP depletion, inhibition of AMPK had no effect on cellular ATP levels. Notably, metabolic stress increased the phosphorylation and activity of Akt. Furthermore, inhibition of AMPK, with CC or gene silencing, abrogated the ability of metabolic stress to activate Akt. The augmentation of apoptosis induced by inhibition of AMPK was comparable to that induced by inhibition of Akt. We conclude that activation of AMPK following acute metabolic stress plays a major role in promoting the viability of cultured proximal tubular cells. Protection by AMPK appears to be due not to AMPK-mediated conservation of cell energy stores, but rather, at least in part, to AMPK-mediated activation of Akt.

Ski negatively regulates erythroid differentiation through its interaction with GATA1.

The Ski oncoprotein dramatically affects cell growth, differentiation, and/or survival. Recently, Ski was shown to act in distinct signaling pathways including those involving nuclear receptors, transforming growth factor beta, and tumor suppressors. These divergent roles of Ski are probably dependent on Ski's capacity to bind multiple partners with disparate functions. In particular, Ski alters the growth and differentiation program of erythroid progenitor cells, leading to malignant leukemia. However, the mechanism underlying this important effect has remained elusive. Here we show that Ski interacts with GATA1, a transcription factor essential in erythropoiesis. Using a Ski mutant deficient in GATA1 binding, we show that this Ski-GATA1 interaction is critical for Ski's ability to repress GATA1-mediated transcription and block erythroid differentiation. Furthermore, the repression of GATA1-mediated transcription involves Ski's ability to block DNA binding of GATA1. This finding is in marked contrast to those in previous reports on the mechanism of repression by Ski, which have described a model involving the recruitment of corepressors into DNA-bound transcription complexes. We propose that Ski cooperates in the process of transformation in erythroid cells by interfering with GATA1 function, thereby contributing to erythroleukemia.

Human adenovirus serotypes 4 and 11 show higher binding affinity and infectivity for endothelial and carcinoma cell lines than serotype 5.

Adenoviruses are promising vectors for human cancer gene therapy. However, the extensively used adenoviruses serotypes 2 and 5 (Ad2 and Ad5) from species C have a major disadvantage in being highly prevalent; thus, most adults have an immunity against the two viruses. Furthermore, the expression of coxsackievirus and adenovirus receptors for Ad2 and Ad5 varies in different cells. This study aims to identify adenovirus serotypes with specific tropism for endothelial cells and epithelial tumour cells. Comparison of the binding affinities of Ad31, Ad11, Ad5, Ad37, Ad4 and Ad41, belonging to species A-F, respectively, to established cell lines of hepatoma (HepG2), breast cancer (CAMA and MG7), prostatic cancer (DU145 and LNCaP) and laryngeal cancer (Hep2), as well as to endothelial cells (HMEC), was carried out by flow cytometric analysis. Ad11 from species B showed markedly higher binding affinity than Ad5 for the endothelial cell line and all carcinoma cell lines studied. Ad4 showed a specific binding affinity for hepatoma cells and laryneal carcinoma cells. The ability of Ad11, Ad4 and Ad5 to be expressed in hepatoma, breast cancer and endothelial cell lines was studied by immunostaining and (35)S-labelling of viral proteins in infected cells. Ad11 and Ad4 manifested a higher proportion of infected cells and a higher degree of hexon expression than Ad5.