Development and Validation of a Simple and Reliable HPLC-UV Method for Determining Gemcitabine Levels: Application in Pharmacokinetic Analysis.

Background and Objectives: Gemcitabine has been used to treat various solid cancers, including, since 1997, metastatic pancreatic cancer. Here, we developed an HPLC-UV method to determine serum gemcitabine levels and use it in pharmacokinetic studies. Materials and Methods: The analysis was performed after a single protein precipitation step on a reversed-phase column, isocratically eluted with sodium phosphate buffer and methanol. For the pharmacokinetic study, NOD/SCID mice received a single dose of gemcitabine at 100 mg/kg by either subcutaneous (SC) or intraperitoneal (IP) administration. Blood samples were collected at 5, 15, and 30 min and 1, 2, 4, and 6 h after the administration of gemcitabine for further analysis. Results: The duration of the analysis was ~12.5 min. The calibration curve was linear (r2 = 0.999) over the range of 1-400 µM. The mean recovery of GEM was 96.53% and the limit of detection was 0.166 µΜ. T1/2, Tmax, Cmax, AUC0-t, and clearance were 64.49 min, 5.00 min, 264.88 µmol/L, 9351.95 µmol/L*min, and 0.0103(mg)/(µmol/L)/min, respectively, for the SC administration. The corresponding values for the IP administration were 59.34 min, 5.00 min, 300.73 µmol/L, 8981.35 µmol/L*min and 0.0108(mg)/(µmol/L)/min (not statistically different from the SC administration). Conclusions: A simple, valid, sensitive, and inexpensive method for the measurement of gemcitabine in serum has been developed. This method may be useful for monitoring gemcitabine levels in cancer patients as part of therapeutic drug monitoring.

A Comparative Analysis of ARX and ANFIS Models for Tumor Growth Prediction Under Single and Multi-agent Chemotherapy.

BACKGROUND/AIM: Despite the advances in oncology and cancer treatment over the past decades, cancer remains one of the deadliest diseases. This study focuses on further understanding the complex nature of cancer by using mathematical tumor modeling to understand, capture as best as possible, and describe its complex dynamics under chemotherapy treatment. MATERIALS AND METHODS: Focusing on autoregressive with exogenous inputs, i.e., ARX, and adaptive neuro-fuzzy inference system, i.e., ANFIS, models, this work investigates tumor growth dynamics under both single and combination anticancer agent chemotherapy treatments using chemotherapy treatment data on xenografted mice. RESULTS: Four ARX and ANFIS models for tumor growth inhibition were developed, estimated, and evaluated, demonstrating a strong correlation with tumor weight data, with ANFIS models showing superior performance in handling the multi-agent tumor growth complexities. These findings suggest potential clinical applications of the ANFIS models through further testing. Both types of models were also tested for their prediction capabilities across different chemotherapy schedules, with accurate forecasting of tumor growth up to five days in advance. The use of adaptive prediction and sliding (moving) data window techniques allowed for continuous model updating, ensuring more robust predictive capabilities. However, long-term forecasting remains a challenge, with accuracy declining over longer prediction horizons. CONCLUSION: While ANFIS models showed greater reliability in predictions, the simplicity and rapid deployment of ARX models offer advantages in situations requiring immediate approximations. Future research with larger, more diverse datasets and by exploring varying model complexities is recommended to improve the models' reliability and applicability in clinical decision-making, thereby aiding the development of personalized chemotherapy regimens.

Targeting of Notch, IL-1, and leptin has therapeutic potential in xenograft colorectal cancer.

Background/aim: Colorectal cancer (CRC) is a fatal malignancy type and its occurence still needs to be explored mechanistically. Notch, IL-1, and leptin crosstalk is reported to play a role in the proliferation, migration, and expression of proangiogenic molecules. In this study, we aimed to investigate the effect of inhibition of Notch, IL-1, and leptin on CRC. Materials and methods: To generate colorectal cancer tumor xenografts, 1 × 107 cells from exponentially growing cultures of HCT-15 cells were injected subcutaneously, at the axillary region of the left and right rear flanks of forty NOD.CB17-Prkdcscid/J (NOD/SCID) female mice. The mice were then monitored for the development of tumors and were randomly divided into five groups when tumor sizes reached a volume of approximately 150 mm3. Mice were used to determine the effectiveness of the gamma-secretase inhibitor (DAPT, Notch inhibitor), the interleukin-1 receptor antagonist (Anakinra) and the leptin receptor antagonist (Allo aca) against tumor growth. The mice were euthanized by CO2 inhalation immediately after the treatments finished, and all efforts were made to minimize suffering. Tumors were excissed for RT-PCR and histological analysis. Results: There is an intact Notch, IL-1, and leptin signaling axis, and in vivo antagonism of Notch, IL-1, and leptin affects mRNA and protein expression of inflammatory and angiogenic molecules. Conclusion: Present data suggest that targeting Notch, IL-1, and leptin may be possesses therapeutic potential in CRC.

Caveolin-1 protein expression as a prognostic biomarker of gastrointestinal tumours: A systematic review and meta-analysis.

BACKGROUND: Gastrointestinal (GI) cancers remain a major threat worldwide, accounting for over 30% of cancer deaths. The identification of novel prognostic biomarkers remains a challenge despite significant advances in the field. The CAV1 gene, encoding the caveolin-1 protein, remains enigmatic in cancer and carcinogenesis, as it has been proposed to act as both a tumour promoter and a tumour suppressor. METHODS: To analyse the differential role of caveolin-1 expression in both tumour cells and stroma in relation to prognosis in GI tumours, we performed a systematic review and meta-analysis according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines; PROSPERO registration number: CRD42022299148. RESULTS: Our analysis showed that high levels of caveolin-1 in tumour cells were associated with poor prognosis and inferior overall survival (OS) in oesophageal and pancreatic cancer and hepatocellular carcinoma (HCC), but not in gastric and colorectal cancer. Importantly, our study showed that higher stromal caveolin-1 expression was associated with significantly longer OS and disease-free survival in colorectal cancer. Analysis of stromal caveolin-1 expression in the remaining tumours showed a similar trend, although it did not reach statistical significance. CONCLUSIONS: The data suggest that caveolin-1 expression in the tumour cells of oesophageal, pancreatic cancer and HCC and in the stroma of colorectal cancer may be an important novel predictive biomarker for the clinical management of these diseases in a curative setting. However, the main conclusion of our analysis is that caveolin-1 expression should always be assessed separately in stroma and tumour cells.

P90 ribosomal S6 kinases: A bona fide target for novel targeted anticancer therapies?

The 90 kDa ribosomal S6 kinase (RSK) family of proteins is a group of highly conserved Ser/Thr kinases. They are downstream effectors of the Ras/ERK/MAPK signaling cascade. ERK1/2 activation directly results in the phosphorylation of RSKs, which further, through interaction with a variety of different downstream substrates, activate various signaling events. In this context, they have been shown to mediate diverse cellular processes like cell survival, growth, proliferation, EMT, invasion, and metastasis. Interestingly, increased expression of RSKs has also been demonstrated in various cancers, such as breast, prostate, and lung cancer. This review aims to present the most recent advances in the field of RSK signaling that have occurred, such as biological insights, function, and mechanisms associated with carcinogenesis. We additionally present and discuss the recent advances but also the limitations in the development of pharmacological inhibitors of RSKs, in the context of the use of these kinases as putative, more efficient targets for novel anticancer therapeutic approaches.

Influence of the Topology of Poly(L-Cysteine) on the Self-Assembly, Encapsulation and Release Profile of Doxorubicin on Dual-Responsive Hybrid Polypeptides.

Τhe synthesis of a series of novel hybrid block copolypeptides based on poly(ethylene oxide) (PEO), poly(l-histidine) (PHis) and poly(l-cysteine) (PCys) is presented. The synthesis of the terpolymers was achieved through a ring-opening polymerization (ROP) of the corresponding protected N-carboxy anhydrides of Nim-Trityl-l-histidine and S-tert-butyl-l-cysteine, using an end-amine-functionalized poly(ethylene oxide) (mPEO-NH2) as macroinitiator, followed by the deprotection of the polypeptidic blocks. The topology of PCys was either the middle block, the end block or was randomly distributed along the PHis chain. These amphiphilic hybrid copolypeptides assemble in aqueous media to form micellar structures, comprised of an outer hydrophilic corona of PEO chains, and a pH- and redox-responsive hydrophobic layer based on PHis and PCys. Due to the presence of the thiol groups of PCys, a crosslinking process was achieved further stabilizing the nanoparticles (NPs) formed. Dynamic light scattering (DLS), static light scattering (SLS) and transmission electron microscopy (TEM) were utilized to obtain the structure of the NPs. Moreover, the pH and redox responsiveness in the presence of the reductive tripeptide of glutathione (GSH) was investigated at the empty as well as the loaded NPs. The ability of the synthesized polymers to mimic natural proteins was examined by Circular Dichroism (CD), while the study of zeta potential revealed the "stealth" properties of NPs. The anticancer drug doxorubicin (DOX) was efficiently encapsulated in the hydrophobic core of the nanostructures and released under pH and redox conditions that simulate the healthy and cancer tissue environment. It was found that the topology of PCys significantly altered the structure as well as the release profile of the NPs. Finally, in vitro cytotoxicity assay of the DOX-loaded NPs against three different breast cancer cell lines showed that the nanocarriers exhibited similar or slightly better activity as compared to the free drug, rendering these novel NPs very promising materials for drug delivery applications.

Pancreatic Cancer Organoids: An Emerging Platform for Precision Medicine?

Despite recent therapeutic advances, pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies, with remarkable resistance to treatment, poor prognosis, and poor clinical outcome. More efficient therapeutic approaches are urgently needed to improve patients' survival. Recently, the development of organoid culture systems has gained substantial attention as an emerging preclinical research model. PDAC organoids have been developed to study pancreatic cancer biology, progression, and treatment response, filling the translational gap between in vitro and in vivo models. Here, we review the rapidly evolving field of PDAC organoids and their potential as powerful preclinical tools that could pave the way towards precision medicine for pancreatic cancer.

Increased Expression of the Mitochondrial Glucocorticoid Receptor Enhances Tumor Aggressiveness in a Mouse Xenograft Model.

Mitochondria are important organelles for cellular physiology as they generate most of the energy requirements of the cell and orchestrate many biological functions. Dysregulation of mitochondrial function is associated with many pathological conditions, including cancer development. Mitochondrial glucocorticoid receptor (mtGR) is proposed as a crucial regulator of mitochondrial functions via its direct involvement in the regulation of mitochondrial transcription, oxidative phosphorylation (OXPHOS), enzymes biosynthesis, energy production, mitochondrial-dependent apoptosis, and regulation of oxidative stress. Moreover, recent observations revealed the interaction of mtGR with the pyruvate dehydrogenase (PDH), a key player in the metabolic switch observed in cancer, indicating direct involvement of mtGR in cancer development. In this study, by using a xenograft mouse model of mtGR-overexpressing hepatocarcinoma cells, we showed increased mtGR-associated tumor growth, which is accompanied by reduced OXPHOS biosynthesis, reduction in PDH activity, and alterations in the Krebs cycle and glucose metabolism, metabolic alterations similar to those observed in the Warburg effect. Moreover, autophagy activation is observed in mtGR-associated tumors, which further support tumor progression via increased precursors availability. Thus, we propose that increased mitochondrial localization of mtGR is associated with tumor progression possible via mtGR/PDH interaction, which could lead to suppression of PDH activity and modulation of mtGR-induced mitochondrial transcription that ends up in reduced OXPHOS biosynthesis and reduced oxidative phosphorylation versus glycolytic pathway energy production, in favor of cancer cells.

Bioinformatic Analysis of the BCL-xL/BCL2L1 Interactome in Patients with Pancreatic Cancer.

Objectives: The aim of the present study was to analyze the differential gene expression of BCL-xL/BCL2L and the associated genetic, molecular, and biologic functions in pancreatic ductal adenocarcinoma (PDAC) by employing advanced bioinformatics to investigate potential candidate genes implicated in the pathogenesis of PDAC. Materials and Methods: Bioinformatic techniques were employed to build the gene network of BCL-xL, to assess the translational profile of BCL-xL in PDAC, assess its role in predicting PDAC, and investigate the associated biologic functions and the regulating miRNA families. Results: Microarray data extracted from one dataset was incorporated, including 130 samples (PDAC: 69; Control: 61). In addition, the expression level of BCL-xL was higher in PDAC compared to control samples (p < 0.001). Furthermore, BCL-xL demonstrated excellent discrimination (AUC: 0.83 [95% Confidence Intervals: 0.76, 0.90]; p < 0.001) and calibration (R squared: 0.31) traits for PDAC. A gene set enrichment analysis (GSEA) demonstrated the molecular functions and miRNA families (hsa-miR-4804-5p, hsa-miR-4776-5p, hsa-miR-6770-3p, hsa-miR-3619-3p, and hsa-miR-7152-3p) related to BCL-xL. Conclusions: The current findings unveil the biological implications of BCL-xL in PDAC and the related molecular functions and miRNA families.

Hydrophilic Random Cationic Copolymers as Polyplex-Formation Vectors for DNA.

Research on the improvement and fabrication of polymeric systems as non-viral gene delivery carriers is required for their implementation in gene therapy. Random copolymers have not been extensively utilized for these purposes. In this regard, double hydrophilic poly[(2-(dimethylamino) ethyl methacrylate)-co-(oligo(ethylene glycol) methyl ether methacrylate] [P(DMAEMA-co-OEGMA)] random copolymers were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The copolymers were further modified by quaternization of DMAEMA tertiary amine, producing the cationic P(QDMAEMA-co-OEGMA) derivatives. Fluorescence and ultraviolet-visible (UV-vis) spectroscopy revealed the efficient interaction of copolymers aggregates with linear DNAs of different lengths, forming polyplexes, with the quaternized copolymer aggregates exhibiting stronger binding affinity. Light scattering techniques evidenced the formation of polyplexes whose size, molar mass, and surface charge strongly depend on the N/P ratio (nitrogen (N) of the amine group of DMAEMA/QDMAEMA over phosphate (P) groups of DNA), DNA length, and length of the OEGMA chain. Polyplexes presented colloidal stability under physiological ionic strength as shown by dynamic light scattering. In vitro cytotoxicity of the empty nanocarriers was evaluated on HEK293 as a control cell line. P(DMAEMA-co-OEGMA) copolymer aggregates were further assessed for their biocompatibility on 4T1, MDA-MB-231, MCF-7, and T47D breast cancer cell lines presenting high cell viability rates.

Establishment of Patient-derived Orthotopic Xenografts (PDX) as Models for Pancreatic Ductal Adenocarcinoma.

BACKGROUND/AIM: Pancreatic cancer (PC) is one of the leading causes of cancer-related death. The purpose of the present study was to establish a patient-derived orthotopic xenograft model (PDOX) for pancreatic ductal adenocarcinoma (PDAC), thus providing a tumor microenvironment resembling that of the human pancreas to identify novel potential biomarkers and treatment regimens. MATERIALS AND METHODS: PDAC tissue samples were received from 35 patients, following informed consent, and three mouse strains were implemented. RESULTS: Successful PDOX engraftment was performed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID gamma (NSG) mice. Nonetheless, we found a higher rate of successful engraftment and tumor growth in NSG compared to NOD/SCID mice, possibly owning to the different level of immunosuppression and more specifically of the natural killer cells presence. CONCLUSION: Our suggested PDOX model represents a preclinical cancer research model with a high affinity for the patient's tumor microenvironment, thus enabling the acceleration of PDAC research.

Effects of combined administration of doxorubicin and chloroquine on lung pathology in mice with solid Ehrlich ascites carcinoma.

Combined use of a chemotherapeutic agent and an autophagy inhibitor is a novel cancer treatment strategy. We investigated the effects of chloroquine (CQ) on lung pathology caused by both solid Ehrlich ascites carcinoma (EAC) and doxorubicin (DXR). A control group and eight experimental groups of adult female mice were inoculated subcutaneously with 2.5 × 106 EAC cells. DXR (1.5 mg/kg and 3 mg/kg) and CQ (25 mg/kg and 50 mg/kg) alone or in combination were injected intraperitoneally on days 2, 7 and 12 following inoculation with EAC cells. Lung tissue samples were examined using immunohistochemistry (IHC) for endothelial (eNOS), inducible nitric oxide synthase (iNOS) and neutrophil gelatinase-associated lipocalin (NGAL). Serum catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured using ELISA. We found decreased levels of iNOS and eNOS in the groups that received 1.5 mg/kg DXR alone and in combination with 25 mg/kg and 50 mg/kg CQ. Combined administration of DXR and CQ partially prevented disruption of alveolar structure. Levels of antioxidant enzymes and MDA were lower in all treated groups; the greatest reduction was observed in mice that received the combination of 25 mg/kg CQ + 1.5 mg/kg DXR. Levels of NGAL were elevated in all treated groups. We found that CQ ameliorated both EAC and DOX induced lung pathology in female mice with solid EAC by reducing oxidative stress.

Prognostic significance and therapeutic implications of Caveolin-1 in gastrointestinal tract malignancies.

Caveolin-1 (CAV1) is expressed in several solid tumors both in cancerous cells as well as in tumor stroma and is reported to be related to cancer progression, metastasis, therapy resistance and clinical outcomes. Many studies report contrasting functions of this protein depending on the tumor cell model, the tumor type, or the stage of cancer studied. This protein is reported to function both as tumor suppressor and as tumor promoter. In this review, we aim to summarize translational and clinical studies that provide evidence of the role of CAV1 in tumor progression and survival outcome focusing on tumors of the gastrointestinal (GI) tract. Towards this aim, a detailed search has been performed for studies on the expression and the role of CAV1 in oesophageal, gastric, colorectal, pancreatic cancer and cholangiocarcinoma prognosis. We also review and discuss the implication of CAV1 in the outcome of pharmacological interventions. We conclude that CAV1 has the potential to become an important prognostic, and possibly predictive, biomarker in GI malignancies. It may also become a novel target towards the development of improved cancer therapies. However, it is obvious that there remains a lack of consensus on important issues such as the methodologies and cut-off levels in caveolin assessment. This ultimately result in many studies being contradictory not only in terms of the role of CAV1 as a tumor-promoting or suppressing gene but also in terms of the tumor compartment in which the levels of this protein may be of clinical significance. Addressing these important technical issues, in conjunction with a further elucidation of the role of CAV1 in tumor formation and progression, will delineate the importance of CAV1 in prognostic and therapeutic perspectives.

Heterodimeric IL-15 (hetIL-15) reduces circulating tumor cells and metastasis formation improving chemotherapy and surgery in 4T1 mouse model of TNBC.

Immunotherapy has emerged as a viable approach in cancer therapy, with cytokines being of great interest. Interleukin IL-15 (IL-15), a cytokine that supports cytotoxic immune cells, has been successfully tested as an anti-cancer and anti-metastatic agent, but combinations with conventional chemotherapy and surgery protocols have not been extensively studied. We have produced heterodimeric IL-15 (hetIL-15), which has shown anti-tumor efficacy in several murine cancer models and is being evaluated in clinical trials for metastatic cancers. In this study, we examined the therapeutic effects of hetIL-15 in combination with chemotherapy and surgery in the 4T1 mouse model of metastatic triple negative breast cancer (TNBC). hetIL-15 monotherapy exhibited potent anti-metastatic effects by diminishing the number of circulating tumor cells (CTCs) and by controlling tumor cells colonization of the lungs. hetIL-15 treatment in combination with doxorubicin resulted in enhanced anti-metastatic activity and extended animal survival. Systemic immune phenotype analysis showed that the chemoimmunotherapeutic regimen shifted the tumor-induced imbalance of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in favor of cytotoxic effector cells, by simultaneously decreasing PMN-MDSCs and increasing the frequency and activation of effector (CD8+T and NK) cells. Tumor resection supported by neoadjuvant and adjuvant administration of hetIL-15, either alone or in combination with doxorubicin, resulted in the cure of approximately half of the treated animals and the development of anti-4T1 tumor immunity. Our findings demonstrate a significant anti-metastatic potential of hetIL-15 in combination with chemotherapy and surgery and suggest exploring the use of this regimen for the treatment of TNBC.

Effects of In Vitro Muscle Contraction on Thermogenic Protein Levels in Co-Cultured Adipocytes.

The crosstalk between the exercising muscle and the adipose tissue, mediated by myokines and metabolites, derived from both tissues during exercise has created a controversy between animal and human studies with respect to the impact of exercise on the browning process. The aim of this study was to investigate whether co-culturing of C2C12 myotubes and 3T3-L1 adipocytes under the stimuli of electrical pulse stimulation (EPS) mimicking muscle contraction can impact the expression of UCP1, PGC-1a, and IL-6 in adipocytes, therefore providing evidence on the direct crosstalk between adipocytes and stimulated muscle cells. In the co-cultured C2C12 cells, EPS increased the expression of PGC-1a (p = 0.129; d = 0.73) and IL-6 (p = 0.09; d = 1.13) protein levels. When EPS was applied, we found that co-culturing led to increases in UCP1 (p = 0.044; d = 1.29) and IL-6 (p = 0.097; d = 1.13) protein expression in the 3T3-L1 adipocytes. The expression of PGC-1a increased by EPS but was not significantly elevated after co-culturing (p = 0.448; d = 0.08). In vitro co-culturing of C2C12 myotubes and 3T3-L1 adipocytes under the stimuli of EPS leads to increased expression of thermogenic proteins. These findings indicate changes in the expression pattern of proteins related to browning of adipose tissue, supporting the use of this in vitro model to study the crosstalk between adipocytes and contracting muscle.

Tumor Chemosensitivity Assays Are Helpful for Personalized Cytotoxic Treatments in Cancer Patients.

Tumor chemosensitivity assays (TCAs), also known as drug response assays or individualized tumor response tests, have been gaining attention over the past few decades. Although there have been strong positive correlations between the results of these assays and clinical outcomes, they are still not considered routine tests in the care of cancer patients. The correlations between the assays' results (drug sensitivity or resistance) and the clinical evaluations (e.g., response to treatment, progression-free survival) are highly promising. However, there is still a need to design randomized controlled prospective studies to secure the place of these assays in routine use. One of the best ideas to increase the value of these assays could be the combination of the assay results with the omics technologies (e.g., pharmacogenetics that gives an idea of the possible side effects of the drugs). In the near future, the importance of personalized chemotherapy is expected to dictate the use of these omics technologies. The omics relies on the macromolecules (Deoxyribonucleic acid -DNA-, ribonucleic acid -RNA-) and proteins (meaning the structure) while TCAs operate on living cell populations (meaning the function). Therefore, wise combinations of TCAs and omics could be a highly promising novel landscape in the modern care of cancer patients.

Chimeric Stimuli-Responsive Liposomes as Nanocarriers for the Delivery of the Anti-Glioma Agent TRAM-34.

Nanocarriers are delivery platforms of drugs, peptides, nucleic acids and other therapeutic molecules that are indicated for severe human diseases. Gliomas are the most frequent type of brain tumor, with glioblastoma being the most common and malignant type. The current state of glioma treatment requires innovative approaches that will lead to efficient and safe therapies. Advanced nanosystems and stimuli-responsive materials are available and well-studied technologies that may contribute to this effort. The present study deals with the development of functional chimeric nanocarriers composed of a phospholipid and a diblock copolymer, for the incorporation, delivery and pH-responsive release of the antiglioma agent TRAM-34 inside glioblastoma cells. Nanocarrier analysis included light scattering, protein incubation and electron microscopy, and fluorescence anisotropy and thermal analysis techniques were also applied. Biological assays were carried out in order to evaluate the nanocarrier nanotoxicity in vitro and in vivo, as well as to evaluate antiglioma activity. The nanosystems were able to successfully manifest functional properties under pH conditions, and their biocompatibility and cellular internalization were also evident. The chimeric nanoplatforms presented herein have shown promise for biomedical applications so far and should be further studied in terms of their ability to deliver TRAM-34 and other therapeutic molecules to glioblastoma cells.

Crocus sativus L. Extract and Its Constituents: Chemistry, Pharmacology and Therapeutic Potential.

Natural products or organic compounds isolated from natural sources as primary or secondary metabolites have inspired numerous drugs [...].