Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance.

Rationale: Growing evidence points to the tumor microenvironment's role in developing drug resistance. A key element of this microenvironment is inter-cellular communication, which includes the release of membrane-encapsulated vesicles containing various cargo, known as extracellular vesicles (EVs). Understanding how EVs contribute to acquired resistance holds significant clinical implications. Methods: Differential centrifugation-based methods were used to isolate EVs from established cell lines and human plasma. TMT labeling proteomics analysis of EVs revealed an abundance of metabolic transporter proteins. Increased expression of SLC1A5 in EVs of patient-derived plasma and cell lines rendered resistant to tyrosine kinase inhibitors and its relationship with progression-free survival was assessed using Kaplan-Meier survival plot. Gene knockdown and overexpression of SLC1A5 were used to validate its effect on Tyrosine kinase inhibitor (TKI) resistance. Co-culture assays using inserts was used to evaluate the effect of resistant EVs on normal fibroblasts and epithelial cells. Next, mouse-derived tumor slices (MDTS) were cultured in vitro to assess the effect of resistant EVs. Results: We report here that TKI-sensitive cells are rendered resistant upon incubation with EVs derived from TKI-resistant cell lines. Metabolic transporters, in particular SLC1A5 and SLC25A5, are upregulated in EVs derived from TKI-resistant cells and plasma from patients harbouring TKI-resistant tumors and in TKI-resistant cell lines. Furthermore, we also provide evidence for the increased abundance of pSTAT3 and the stemness marker ALDH1A1 upon EV-induced resistance. Notably, resistant EVs trigger phenotypic and functional switching of lung-derived fibroblasts into tumor-associated fibroblasts, significantly increasing their migratory and invasive capacities. Conclusions: Our findings support the role of metabolic transporters within tumor-derived EVs in reshaping the tumor microenvironment to promote therapy resistance, which could have potential diagnostic, prognostic, and therapeutic implications.

Inhibitory Potential of Resveratrol in Cancer Metastasis: From Biology to Therapy.

Cancer metastasis is a significant challenge in cancer treatment, and most existing drugs are designed to inhibit tumor growth but are often ineffective in treating metastatic cancer, which is the leading cause of cancer-related deaths. Resveratrol, a polyphenol found in grapes, berries, and peanuts, has shown potential in preclinical studies as an anticancer agent to suppress metastasis. However, despite positive results in preclinical studies, little progress has been made in clinical trials. To develop resveratrol as an effective anticancer agent, it is crucial to understand its cellular processes and signaling pathways in tumor metastasis. This review article evaluates the current state and future development strategies of resveratrol to enhance its potency against cancer metastasis within its therapeutic dose. In addition, we critically evaluate the animal models used in preclinical studies for cancer metastasis and discuss novel techniques to accelerate the translation of resveratrol from bench to bedside. The appropriate selection of animal models is vital in determining whether resveratrol can be further developed as an antimetastatic drug in cancer therapy.

FAM3C in circulating tumor-derived extracellular vesicles promotes non-small cell lung cancer growth in secondary sites.

Rationale: Metastasis is a complex process with a molecular underpinning that remains unclear. We hypothesize that cargo proteins conducted by extracellular vesicles (EVs) released from tumors may confer growth and metastasis potential on recipient cells. Here, we report that a cytokine-like secreted protein, FAM3C, contributes to late-stage lung tumor progression. Methods: EV protein profiling was conducted with an unbiased proteomic mass spectrometry analysis on non-small cell lung cancer (NSCLC) and normal lung fibroblast cell lines. Expression of FAM3C was confirmed in a panel of NSCLC cell lines, and correlated to the invasive and metastatic potentials. Functional phenotype of endogenous FAM3C and tumor-derived EVs (TDEs) were further investigated using various biological approaches in RNA and protein levels. Metastasis potential of TDEs secreted by FAM3C-overexpressing carcinoma cells was validated in mouse models. Results: Transcriptomic meta-analysis of pan-cancer datasets confirmed the overexpression of FAM3C - a gene encoding for interleukin-like EMT inducer (ILEI) - in NSCLC tumors, with strong association with poor patient prognosis and cancer metastasis. Aberrant expression of FAM3C in lung carcinoma cells enhances cellular transformation and promotes distant lung tumor colonization. In addition, higher FAM3C concentrations were detected in EVs extracted from plasma samples of NSCLC patients compared to those of healthy subjects. More importantly, we defined a hitherto-unknown mode of microenvironmental crosstalk involving FAM3C in EVs, whereby the delivery and uptake of FAM3C via TDEs enhances oncogenic signaling - in recipient cells that phenocopies the cell-endogenous overexpression of FAM3C. The oncogenicity transduced by FAM3C is executed via a novel interaction with the Ras-related protein RalA, triggering the downstream activation of the Src/Stat3 signaling cascade. Conclusions: Our study describes a novel mechanism for FAM3C-driven carcinogenesis and shed light on EV FAM3C as a driver for metastatic lung tumors that could be exploited for cancer therapeutics.

FBXW5 Promotes Tumorigenesis and Metastasis in Gastric Cancer via Activation of the FAK-Src Signaling Pathway.

: F-box/WD repeat-containing protein 5 (FBXW5) is a member of the FBXW subclass of F-box proteins. Despite its known function as a component of the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex, the role of FBXW5 in gastric cancer tumorigenesis and metastasis has not been investigated. The present study investigates the role of FBXW5 in tumorigenesis and metastasis, as well as the regulation of key signaling pathways in gastric cancer; using in-vitro FBXW5 knockdown/overexpression cell line and in-vivo models. In-vitro knockdown of FBXW5 results in a decrease in cell proliferation and cell cycle progression, with a concomitant increase in cell apoptosis and caspase-3 activity. Furthermore, knockdown of FBXW5 also leads to a down regulation in cell migration and adhesion, characterized by a reduction in actin polymerization, focal adhesion turnover and traction forces. This study also delineates the mechanistic role of FBXW5 in oncogenic signaling as its inhibition down regulates RhoA-ROCK 1 (Rho-associated protein kinase 1) and focal adhesion kinase (FAK) signaling cascades. Overexpression of FBXW5 promotes in-vivo tumor growth, whereas its inhibition down regulates in-vivo tumor metastasis. When considered together, our study identifies the novel oncogenic role of FBXW5 in gastric cancer and draws further interest regarding its clinical utility as a potential therapeutic target.

A Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Nimbolide in Mouse Serum: Application to a Preclinical Pharmacokinetics Study.

A sensitive and robust liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of nimbolide in mouse serum. Exemestane was used as the internal standard (IS). Here, we employed acetonitrile-based protein precipitation (PPT) for serum sample preparation, and performed chromatographic separation using an ODS Hypersil C18 column (100 mm × 2.1 mm, 5 µm) with gradient elution (0.1% formic acid in water vs 100% acetonitrile). The run time was 6 min. Instrumental analysis was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the multiple-reaction monitoring (MRM) under positive mode. A good linear calibration was achieved in the 5⁻1000 ng/mL range. The intra- and inter-day precisions for nimbolide were ≤12.6% and ≤13.9% respectively. Intra-day accuracy ranged from 96.9⁻109.3%, while inter-day accuracy ranged from 94.3⁻110.2%. The matrix effect of nimbolide, detected but consistent at low and high concentrations, do not affect linearity of standard curve. In conclusion, we have developed and validated a sensitive analytical method for determination of a novel natural compound nimbolide in mouse serum, and it has been successfully applied to our preclinical study in investigating the pharmacokinetic properties of nimbolide, which could greatly facilitate the preclinical development of the promising lead compound for anticancer therapy.

Anti-tumor efficacy of Selinexor (KPT-330) in gastric cancer is dependent on nuclear accumulation of p53 tumor suppressor.

Exportin-1 (XPO1) controls the nucleo-cytoplasmic trafficking of several key growth regulatory and tumor suppressor proteins. Nuclear export blockade through XPO1 inhibition is a target for therapeutic inhibition in many cancers. Studies have suggested XPO1 upregulation as an indicator of poor prognosis in gastric cancer. In the current study, we investigated the anti-tumor efficacy of selective inhibitors of nuclear export (SINE) compounds KPT-185, KTP-276 and clinical stage selinexor (KPT-330) in gastric cancer. XPO1 was found to be overexpressed in gastric cancer as compared to adjacent normal tissues and was correlated with poor survival outcomes. Among the 3 SINE compounds, in vitro targeting of XPO1 with selinexor resulted in greatest potency with significant anti-proliferative effects at nano molar concentrations. XPO1 inhibition by selinexor resulted in nuclear accumulation of p53, causing cell cycle arrest and apoptosis. Also, inhibition of XPO1 lead to the cytoplasmic retention of p21 and suppression of survivin. Orally administered selienxor caused significant inhibition of tumor growth in xenograft models of gastric cancer. Furthermore, combination of selinexor with irinotecan exhibited greater anti-tumor effect compared to individual treatment. Taken together, our study underscores the therapeutic utility of XPO1 targeting in gastric cancer and suggests the potential benefits of XPO1 inhibition in-combination with chemotherapy.

Pan-HDAC inhibition by panobinostat mediates chemosensitization to carboplatin in non-small cell lung cancer via attenuation of EGFR signaling.

Accumulating evidence has implicated the aberrant regulation of histone deacetylases (HDACs) as a nexus for multiple cancer hallmarks and in mediating tumor adaptation and resistance to genotoxic chemotherapy, suggesting a rational pairing of HDAC inhibitors with DNA damaging chemotherapeutic agents in the treatment of human malignancies. Here we report that panobinostat (LBH589), a potent pan-HDAC inhibitor, effectively curbed the proliferation of non-small cell lung cancer (NSCLC) cell lines A549, Calu-1, H226, H460, H838 and SKMES-1 at IC50 concentrations between 4 and 31 nmol/L via pleiotropic mechanisms, including crosstalk with EGFR signal transduction cascades. Combination therapy with carboplatin elicited rapid tumor cell kill and effectively restrained anchorage-independent clonogenic survival to a considerably greater extent over either monotherapy. The administration of carboplatin and panobinostat at clinically relevant doses to NOD-SCID xenograft mice drastically stalled disease progression by 92% as compared with negative control (P = .0026), which was greater than the 28% and 54% achieved with either carboplatin (P = .220) or panobinostat (P = .017) alone. These data demonstrate that panobinostat has strong anti-NSCLC activity and chemosensitizes tumors to carboplatin, thus justifying further evaluation of this combination approach in clinical trials.

A novel combinatorial strategy using Seliciclib(®) and Belinostat(®) for eradication of non-small cell lung cancer via apoptosis induction and BID activation.

With conventional anticancer agents for non-small cell lung cancer (NSCLC) reaching therapeutic ceiling, the novel combination using histone deacetylase inhibitor, PXD101 (Belinostat(®)), and CDK inhibitor, CYC202 (Seliciclib(®)), was investigated as an alternative anticancer strategy. At clinically achievable concentration of CYC202 (15 µM), combination therapy resulted in significant reduction in cell proliferation (IC50 = 3.67 ± 0.80 µM, p < 0.05) compared with PXD101 alone (IC50 = 6.56 ± 0.42 µM) in p53 wild-type A549 cells. Significant increase in apoptosis that occurred independently of cell cycle arrest was observed after concurrent treatment. This result was corroborated by greater formation of cleaved caspase-8, caspase-3 and PARP. Up-regulation of p53 and truncated BID protein levels was seen while Mcl-1 and XIAP protein levels were down-regulated upon combined treatment. Further analysis of apoptotic pathways revealed that caspase inhibitors, but not p53 silencing, significantly abrogated the cytotoxic enhancement. Moreover, the enhanced efficacy of this combination was additionally confirmed in p53 null H2444 cells, suggesting the potential of this combination for treatment of NSCLC that are not amenable to effects of conventional p53-inducing agents.

Anticancer properties of nimbolide and pharmacokinetic considerations to accelerate its development.

Nimbolide is one of the main components in the leaf extract of Azadirachta indica (A. indica). Accumulating evidence from various in vitro and in vivo studies indicates that nimbolide possesses potent anticancer activity against several types of cancer and also shows potential chemopreventive activity in animal models. The main mechanisms of action of nimbolide include anti-proliferation, induction of apoptosis, inhibition of metastasis and angiogenesis, and modulation of carcinogen-metabolizing enzymes. Although multiple pharmacodynamic (PD) studies have been carried out, nimbolide is still at the infant stage in the drug development pipeline due to the lack of systematic pharmacokinetic (PK) studies and long-term toxicological studies. Preclinical PK and toxicological studies are vital in determining the dosage range to support the safety of nimbolide for first-in-human clinical trials. In this review, we will provide a comprehensive summary for the current status of nimbolide as an anticancer and chemopreventive lead compound, and highlight the importance of systematic preclinical PK and toxicological studies in accelerating the process of application of nimbolide as a therapeutic agent against various malignancies.

UGT1A1 Mediated Drug Interactions and its Clinical Relevance.

BACKGROUND: The administration of multiple drugs for the treatment of diseases is an integral aspect of modern medicine. Though its purpose is to create the intended therapeutic effect, the unintended consequences of drug interactions can cause severe side effects and subsequent economic losses. Likewise, herbal extracts and supplements with pharmacologically active moieties also have the potential to interact with medications. There are many possible mechanisms on how these moieties could potentially interact, one of which is mediated by modulation of the activity of metabolizing enzymes. One such enzyme of high clinical significance is uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1). Genetic polymorphism of UGT1A1 has been found to affect the plasma concentrations of many drugs, and may even be linked to treatment outcome. OBJECTIVE: This mini-review summarized the potential in vitro and in vivo interactions mediated by UGT1A1. METHOD: Firstly, literature search was conducted using the Web of Knowledge database. No date limitation was applied to the search. Following which, the interactions were stratified into 3 main categories based on its clinical significance. Both herbal and pharmacological drug moieties are covered within the scope of this mini-review. RESULTS: Of 35 UGT1A1 induced drug interactions, likely and unlikely to be clinically significant interactions are 11 and 6 respectively. The rest of them are inconclusive. CONCLUSION: We hope that this secondary literature can broaden and update the perspective of clinicians, pharmacists and academics on the interactions mediated by UGT1A1.

Validation of a rapid and sensitive LC-MS/MS method for determination of exemestane and its metabolites, 17ß-hydroxyexemestane and 17ß-hydroxyexemestane-17-O-ß-D-glucuronide: application to human pharmacokinetics study.

A novel, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the evaluation of exemestane pharmacokinetics and its metabolites, 17ß-dihydroexemestane (active metabolite) and 17ß-dihydroexemestane-17-O-ß-D-glucuronide (inactive metabolite) in human plasma. Their respective D3 isotopes were used as internal standards. Chromatographic separation of analytes was achieved using Thermo Fisher BDS Hypersil C18 analytic HPLC column (100 × 2.1 mm, 5 µm). The mobile phase was delivered at a rate of 0.5 mL/min by gradient elution with 0.1% aqueous formic acid and acetonitrile. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionisation (ESI) and monitored by multiple reaction monitoring (MRM) in positive mode. Mass transitions 297 > 121 m/z, 300 > 121 m/z, 299 > 135 m/z, 302 > 135 m/z, 475 > 281 m/z, and 478 > 284 m/z were monitored for exemestane, exemestane-d3, 17ß-dihydroexemestane, 17ß-dihydroexemestane-d3, 17ß-dihydroexemestane-17-O-ß-D-glucuronide, and 17ß-dihydroexemestane-17-O-ß-D-glucuronide-d3 respectively. The assay demonstrated linear ranges of 0.4-40.0 ng/mL, for exemestane; and 0.2-15.0 ng/mL, for 17ß-dihydroexemestane and 17ß-dihydroexemestane-17-O-ß-D-glucuronide, with coefficient of determination (r2) of > 0.998. The precision (coefficient of variation) were ≤10.7%, 7.7% and 9.5% and the accuracies ranged from 88.8 to 103.1% for exemestane, 98.5 to 106.1% for 17ß-dihydroexemestane and 92.0 to 103.2% for 17ß-dihydroexemestane-17-O-ß-D-glucuronide. The method was successfully applied to a pharmacokinetics/dynamics study in breast cancer patients receiving exemestane 25 mg daily orally. For a representative patient, 20.7% of exemestane in plasma was converted into 17ß-dihydroexemestane and 29.0% of 17ß-dihydroexemestane was inactivated as 17ß-dihydroexemestane-17-O-ß-D-glucuronide 24 hours after ingestion of exemestane, suggesting that altered 17-dihydroexemestane glucuronidation may play an important role in determining effect of exemestane against breast cancer cells.

Quantification of L-ergothioneine in human plasma and erythrocytes by liquid chromatography-tandem mass spectrometry.

A sensitive analytical method has been developed and validated for the quantification of L-ergothioneine in human plasma and erythrocytes by liquid chromatography-tandem mass spectrometry. A commercially available isotope-labeled L-ergothioneine-d9 is used as the internal standard. A simple protein precipitation with acetonitrile is utilized for bio-sample preparation prior to analysis. Chromatographic separation of L-ergothioneine is conducted using gradient elution on Alltime C18 (150 mm × 2.1 mm, 5 µ). The run time is 6 min at a constant flow rate of 0.45 ml/min. The mass spectrometer is operated under a positive electrospray ionization condition with multiple reaction monitoring mode. The mass transitions of L-ergothioneine and L-ergothioneine-d9 are m/z 230 > 127 and m/z 239 > 127, respectively. Excellent linearity [coefficient of determination (r(2)) ≥ 0.9998] can be achieved for L-ergothioneine quantification at the ranges of 10 to 10,000 ng/ml, with the intra-day and inter-day precisions at 0.9-3.9% and 1.3-5.7%, respectively, and the accuracies for all quality control samples between 94.5 and 101.0%. This validated analytical method is suitable for pharmacokinetic monitoring of L-ergothioneine in human and erythrocytes. Based on the determination of bio-samples from five healthy subjects, the mean concentrations of L-ergothioneine in plasma and erythrocytes are 107.4 ± 20.5 ng/ml and 1285.0 ± 1363.0 ng/ml, respectively.

Glucuronidation by UGT1A1 is the dominant pathway of the metabolic disposition of belinostat in liver cancer patients.

UNLABELLED: Belinostat is a hydroxamate class HDAC inhibitor that has demonstrated activity in peripheral T-cell lymphoma and is undergoing clinical trials for non-hematologic malignancies. We studied the pharmacokinetics of belinostat in hepatocellular carcinoma patients to determine the main pathway of metabolism of belinostat. The pharmacokinetics of belinostat in liver cancer patients were characterized by rapid plasma clearance of belinostat with extensive metabolism with more than 4-fold greater relative systemic exposure of major metabolite, belinostat glucuronide than that of belinostat. There was significant interindividual variability of belinostat glucuronidation. The major pathway of metabolism involves UGT1A1-mediated glucuronidation and a good correlation has been identified between belinostat glucuronide formation and glucuronidation of known UGT1A1 substrates. In addition, liver microsomes harboring UGT1A1*28 alleles have lower glucuronidation activity for belinostat compared to those with wildtype UGT1A1. The main metabolic pathway of belinostat is through glucuronidation mediated primarily by UGT1A1, a highly polymorphic enzyme. The clinical significance of this finding remains to be determined. TRIAL REGISTRATION: ClinicalTrials.gov NCT00321594.

Method development and validation for rapid quantification of hydroxychloroquine in human blood using liquid chromatography-tandem mass spectrometry.

A novel and specific liquid chromatography-tandem mass spectrometric method (LC-MS/MS) was developed and validated for the quantification of hydroxychloroquine in human blood using its stable labeled isotope, hydroxychloroquine-d4 as the internal standard. Chromatographic separation of analytes was achieved using an Agilent ZORBAX Eclipse XDB - C8 analytical HPLC column (50 mm × 2.1 mm, 5 µm). The mobile phase comprising water containing 0.1% formic acid-acetonitrile (94:6, v/v) was delivered isocratically at a flow rate of 0.5 mL/min. The column effluent was detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) and monitored by multiple reaction monitoring with positive mode. The precursor to product ion transitions of m/z 336 → 247 and m/z 340 → 251 were used to measure the analyte and IS, respectively. The assay demonstrated a good linear dynamic range of 5-2000 ng/mL for hydroxychloroquine in human blood, with coefficient of determination (r(2)) of =0.9999. The values for intra-day and inter-day precisions of hydroxychloroquine were ≤ 7.86% with the accuracies ranged from 93.8% to 107.6%. The chromatographic run time was 3 min, making it possible to achieve a high throughput analysis. This method was used as a bio-analytical tool in a phase I clinical trial to quantify blood hydroxychloroquine concentrations in patients with non-small cell lung cancer receiving both hydroxychloroquine and gefitinib in their treatment.

Rapid determination of gefitinib and its main metabolite, O-desmethyl gefitinib in human plasma using liquid chromatography-tandem mass spectrometry.

A novel, rapid and specific liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous quantification of gefitinib and its predominant metabolite, O-desmethyl gefitinib in human plasma. Chromatographic separation of analytes was achieved on an Alltima C18 analytical HPLC column (150 mm × 2.1 mm, 5 µm) using an isocratic elution mode with a mobile phase comprised acetonitrile and 0.1% formic acid in water (30:70, v/v). The flow rate was 300 µL/min. The chromatographic run time was 3 min. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) in positive mode. Linearity was demonstrated in the range of 5-1000 ng/mL for gefitinib and 5-500 ng/mL for O-desmethyl gefitinib. The intra- and inter-day precisions for gefitinib and O-desmethyl gefitinib were ≤10.8% and the accuracies ranged from 89.7 to 104.7% for gefitinib and 100.4 to 106.0% for O-desmethyl gefitinib. This method was used as a bioanalytical tool in a phase I clinical trial to investigate the possible effect of hydroxychloroquine on the pharmacokinetics of gefitinib. The results of this study enabled clinicians to ascertain the safety of the combination therapy of hydroxychloroquine and gefitinib in patients with advanced (Stage IIIB-IV) non-small cell lung cancer (NSCLC).

Simultaneous determination of raltegravir and raltegravir glucuronide in human plasma by liquid chromatography-tandem mass spectrometric method.

Raltegravir is a highly efficacious inhibitor of HIV integrase. Large pharmacokinetic variability has been reported in clinical trials and this could be due to glucuronidation of raltegravir, the only reported metabolism pathway. In order to precisely evaluate and monitor the raltegravir and raltegravir glucuronide simultaneously, a novel, sensitive and robust liquid chromatography-tandem mass spectrometric method was developed and validated for simultaneous determination of raltegravir and raltegravir glucuronide in human plasma. A simple protein precipitation with acetonitrile was utilized for plasma sample preparation prior to analysis. Baseline chromatographic separation was achieved on a ZORBAX Eclipse XDB-C8 using gradient elution mode. The run time was 9 min at a constant flow rate of 0.4 ml/min. The mass spectrometer was operated under a positive electrospray ionization condition. Excellent linearity (r(2) ≥ 0.9997) was achieved for raltegravir and raltegravir glucuronide in the range of 2-2000 nmol/l. The average recovery of raltegravir and raltegravir glucuronide was 105.8% and 102.2%, respectively. The precision (coefficient of variation) was 1.6-6.6% for raltegravir and 2.1-6.9 for raltegravir glucuronide, respectively. The accuracy was 98.6-106.1% for raltegravir and 96.3-100.3% for raltegravir glucuronide. The plasma samples were tested to be stable after nine freeze-thaw cycles and exposure to room temperature for 24 h. This well-validated assay was applied for the quantification of raltegravir and raltegravir glucuronide in plasma samples within 24 h after a single oral dose of 400 mg raltegravir in six healthy subjects.