Cepharanthine combined with 5-fluorouracil inhibits the growth of p53-mutant human colorectal cancer cells.

Mutant p53 is primarily responsible for ineffectiveness of many anticancer drugs. The present study showed that cepharanthine alone or combined with 5-fluorouracil effectively controlled the growth of HT-29 human colorectal cancer cells harboring mutant p53 both in vitro and in vivo. The combination of cepharanthine and 5-fluorouracil additively induced apoptotic and necrotic cell death. Their combination significantly upregulated the expression of BAK and cleaved PARP in tumor tissues. Moreover, cepharanthine could prevent 5-fluorouracil-induced BCRP and MRP1 expression. These findings suggest that cepharanthine is a promising agent for treating patients with colorectal cancer containing p53 mutation.

Mechanism of Cepharanthine Cytotoxicity in Human Ovarian Cancer Cells.

Cepharanthine (CEP), a medicinal product derived from Stephania cephalantha Hayata, possesses a potent cytotoxicity against several types of cancers. Recently, we have found that CEP could efficiently inhibit the growth of mutated p53 colon cancer cells, which are often resistant to commonly used chemotherapeutic agents. In this study, we evaluated the cytotoxic effect and the underlying mechanisms of CEP on both chemosensitive CaOV-3 and chemoresistant OVCAR-3 ovarian cancer cell lines. The present study demonstrated that CEP significantly inhibited the growth of CaOV-3 and OVCAR-3 cells in a time- and concentration-dependent manner. CEP arrested CaOV-3 and OVCAR-3 cells in the G1 phase and S phase of cell cycle, respectively. Western blot analysis demonstrated that CEP markedly increased the expression of p21Waf1 protein and decreased the expression of cyclins A and D proteins in both CaOV-3 and OVCAR-3 cells. Additionally, CEP triggered apoptotic cell death in OVCAR-3 cells. Taken together, the above results suggest that CEP is a promising anticancer drug for ovarian cancer.

The prominent impairment of liver/intestinal cytochrome P450 and intestinal drug transporters in sepsis-induced acute kidney injury over acute and chronic renal ischemia, a mouse model comparison.

Drug dosing adjustment in sepsis-induced acute kidney injury (sepsis-AKI) is currently adjusted based on renal function. Sepsis is a multiorgan injury, and thus, drug metabolism in sepsis-AKI might be interfered by non-renal factors such as changes in functions of drug-metabolizing enzymes in the liver and functions of intestinal drug transporters. We compared the defect on mouse CYP3A11 (human CYP3A4 representative) in liver and intestine along with several intestinal drug transporters (MDR1a, MRP2, and OATP3) in three mouse models; chronic ischemic reperfusion injury (Chr I/R; 4-week), acute ischemic reperfusion injury (Acute I/R; 24-h), and cecal ligation and puncture (CLP; 24-h) as representative of sepsis-AKI. Decreased expression of CYP3A11 and drug transporters was demonstrated in all models. Among these models, sepsis-AKI had the least severe renal injury (increased BUN and Scr) with the most severe liver injury (increased ALT and changes in liver histopathology), the most severe intestinal leakage (increased serum (1→3)-ß-D-glucan) and the highest increase in serum IL-6. A reduced expression and activity of liver and intestinal CYP3A11 along with intestinal efflux-drug transporter expressions (MDR1a and MRP2), but not drug uptake transporter (OATP3), was predominant in sepsis-AKI compared with acute I/R. Additionally, a reduction of CYP3A4 expression with IL-6 was demonstrated on HepG2 cells implying a direct injury of IL-6 on human liver cells. Differences in drug metabolism were reported between sepsis-AKI and ischemic-AKI confirming that drug dosing adjustment in sepsis-AKI depends not just only on renal function but also on several non-renal factors. Further studies are warranted.

Evaluation of the HC-04 cell line as an in vitro model for mechanistic assessment of changes in hepatic cytochrome P450 3A during adenovirus infection.

HC-04 cells were evaluated as an in vitro model for mechanistic study of changes in the function of hepatic CYP3A during virus infection. Similar to in vivo observations, infection with a first generation recombinant adenovirus significantly inhibited CYP3A4 catalytic activity in an isoform-specific manner. Virus (MOI 100) significantly reduced expression of the retinoid X receptor (RXR) by 30% 96 hours after infection. Cytoplasmic concentrations of the pregnane X receptor (PXR) were reduced by 50%, whereas the amount of the constitutive androstane receptor (CAR) in the nuclear fraction doubled with respect to uninfected controls. Hepatocyte nuclear factor 4α (HNF-4α) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) were also reduced by ∼70% during infection. Virus suppressed CYP3A4 activity in the presence of the PXR agonist rifampicin and did not affect CYP3A4 activity in the presence of the CAR agonist CITCO [6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime], suggesting that virus-induced modification of PXR may be responsible for observed changes in hepatic CYP3A4. The HC-04 cell line is easy to maintain, and CYP3A4 in these cells was responsive to known inducers and suppressors. Dexamethasone (200 µM) and phenobarbital (500 µM) increased activity by 230 and 124%, whereas ketoconazole (10 µM) and lipopolysaccharide (LPS) (10 µg/ml) reduced activity by 90 and 92%, respectively. This suggests that HC-04 cells can be a valuable tool for mechanistic study of drug metabolism during infection and for routine toxicological screening of novel compounds prior to use in the clinic.

Differential effects of montelukast and zafirlukast on MDA­MB­231 triple­negative breast cancer cells: Cell cycle regulation, apoptosis, autophagy, DNA damage and endoplasmic reticulum stress.

Montelukast and zafirlukast, cysteinyl leukotriene receptor antagonists (LTRAs), trigger apoptosis and inhibit cell proliferation of triple­negative breast cancer MDA­MB­231 cells. By contrast, only zafirlukast induces G0/G1 cell cycle arrest. The present study compared the effects of these drugs on proteins regulating cell proliferation, apoptosis, autophagy, and endoplasmic reticulum (ER) and oxidative stress using reverse transcription­quantitative PCR, western blotting and flow cytometry. The expression of proliferating markers, Ki­67 and proliferating cell nuclear antigen, was decreased by both drugs. Zafirlukast, but not montelukast, decreased the expression of cyclin D1 and CDK4, disrupting progression from G1 to S phase. Zafirlukast also increased the expression of p27, a cell cycle inhibitor. Both drugs decreased the expression of anti­apoptotic protein Bcl­2 and ERK1/2 phosphorylation, and increased levels of the autophagy marker LC3­II and DNA damage markers, including cleaved PARP­1, phosphorylated (p)­ATM and p­histone H2AX. The number of caspase 3/7­positive cells was greater in montelukast­treated cells compared with zafirlukast­treated cells. Montelukast induced higher levels of the ER stress marker CHOP compared with zafirlukast. Montelukast activated PERK, activating transcription factor 6 (ATF6) and inositol­requiring enzyme type 1 (IRE1) pathways, while zafirlukast only stimulated ATF6 and IRE1 pathways. GSK2606414, a PERK inhibitor, decreased apoptosis mediated by montelukast, but did not affect zafirlukast­induced cell death. The knockdown of CHOP by small interfering RNA reduced apoptosis triggered by montelukast and zafirlukast. In conclusion, the effects on cell cycle regulator proteins may contribute to cell cycle arrest caused by zafirlukast. The greater apoptotic effects of montelukast may be caused by the higher levels of activated caspase enzymes and the activation of three pathways of ER stress: PERK, ATF6, and IRE1.

Two new rotenoid glycosides from the rhizomes of Stemona curtisii Hook. f.

Two new rotenoid glycosides named stemonal 11-O-ß-D-glucopyranoside and 6-O-methylstemonal 11-O-ß-D-glucopyranoside together with ten known metabolites were isolated from the rhizomes of Stemona curtisii. The chemical structures of the new compounds were elucidated based on the analysis of their 1D and 2D NMR and HRESIMS, while the sugar unit and absolute configuration were determined by chemical hydrolysis and ECD analysis. Among the tested compounds for anti-α-glucosidase assay, stemonal showed an inhibitory effect (IC50 = 38.67 µM), which is 2.4-fold more potent than acarbose. Cytotoxic evaluation against the lung adenocarcinoma A549 cell line indicated that none of the compounds were strongly active to suppress the cancer cell growth at 100 µM. This work describes the occurrence of rotenoids bearing a sugar moiety, which are reported for the first time in the genus Stemona. The isolated compound's α-glucosidase inhibitory potential provides insight for further investigation of natural rotenoids as anti-diabetic agents.

Allyl ether of mansonone G as a potential anticancer agent for colorectal cancer.

Mansonone G (MG), a 1,2-naphthoquinone isolated from the heartwood of Mansonia gagei Drumm, exhibited several pharmacological activities such as anti-bacterial, anti-estrogenic and anti-adipogenic effect. This study evaluated the cytotoxicity of MG and its derivatives as well as determined the mechanism(s) underlying the cytotoxic activity of the most potent MG derivative on two CRC cell lines, HCT-116 cells carrying p53 wild-type and HT-29 cells carrying p53 mutant. We found that MG and its derivatives could inhibit viability of HCT-116 and HT-29 cells in a concentration-dependent manner. Of all semi-synthetic derivatives of MG, allyl ether mansonone G (MG7) was the most potent cytotoxic agent toward cancer cells and less toxic to normal cells. MG7 could induce ROS generation which was associated with cytotoxicity and apoptosis in both HCT-116 and HT-29 cells. Western blot analysis revealed that MG7 downregulated the expression of Bcl-2 and Bcl-xL proteins in both CRC cell lines and upregulated the expression of BAK protein in HT-29 cells. Moreover, MG7 inhibited AKT signaling pathway in both CRC cell lines and modulated ERK1/2 signaling pathway by inhibiting ERK1/2 phosphorylation in HCT-116 cells and activating ERK1/2 phosphorylation in HT-29 cells. Molecular docking revealed that MG7 could bind to the ATP-binding pocket of AKT and ERK1 via hydrophobic interactions.

A new neolignan and a new phenolic acid from the heartwood of Mansonia gagei J.R. Drumm.

One new neolignan (1) and one new phenolic compound (2), together with four known compounds (3-6) were isolated from the heartwood of Mansonia gagei. Their structures were elucidated by extensive spectroscopic analyses, including 1D and 2D NMR and HRESIMS. The absolute configuration of 2 was established based on the DP4+ protocol and by comparison of experimental and calculated ECD spectra. All isolated compounds were evaluated by DPPH assay for antioxidant activity, while compounds 3-6 were assayed using the MTT-based colorimetric assay for cytotoxicity against lung cancer cell line A549. In terms of antioxidant activity, 1 and 3 exhibited stronger activity (IC50 14.91 ± 1.10 and 17.46 ± 0.16 µM, respectively) than the positive control, ascorbic acid (IC50 30.20 ± 0.47 µM). Among the compounds tested for cytotoxicity, compound 3 showed the highest activity, with an IC50 value of 26.04 ± 2.95 µM.

Species differences in the pharmacology and toxicology of PEGylated helper-dependent adenovirus.

Clinically relevant doses of helper-dependent adenoviruses (HDAds) provoke the host response against capsid proteins in primates and rodents. To determine if PEGylation truly affects this, baboons and mice were given either HDAd or PEG-HDAd expressing beta-galactosidase at 5 × 10¹¹ or 3 × 10¹² virus particles per kilogram (vp/kg) by iv infusion. Serum cytokines and blood chemistries were assessed for 96 h. PEG-HDAd reduced IL-6 6-fold in mice and 3-fold in the primate. This vector reduced IL-12 by 50% in both animal models. PEGylation reduced serum transaminases by approximately 50% at each dose in the primate and the mouse. PEGylation did not alter hepatic transduction efficiency in the mouse but did reduce transduction efficiency in the liver and the spleen of primates. Unmodified and PEGylated virus suppressed hepatic CYP3A activity in both animal models. PEGylation doubled the half-life (t(½)) of the virus in the mouse and cut plasma clearance (CL) in half without affecting the half-life in primates. These results suggest that there are notable species-specific differences in the biodistribution of and response to PEG-modified vectors which may be linked to differences in binding properties to coagulation factors, receptor density and tissue architecture in the liver.

Characterization of a nanoparticulate exopolysaccharide from Leuconostoc holzapfelii KM01 and its potential application in drug encapsulation.

Fermentation of Lactic Acid Bacteria (LAB) is considered to be a sustainable approach for polysaccharide production. Herein, exopolysaccharide (EPS)-producing LAB strain KM01 was isolated from Thai fermented dessert, Khao Mak, which was then identified as Leuconostoc holzapfelii. High-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectroscopy suggested that the KM01 EPS comprises α-1,6-linked glucosides. The molecular weight of KM01 EPS was around 500 kDa, but it can form large aggregates formation (MW > 2000 kDa) in an aqueous solution, judged by transmission electron microscopy and dynamic light scattering to be around 150 nm in size. Furthermore, this KM01 EPS form highly viscous hydrogels at concentrations above 5% (w/v). The formation of hydrogels and nanoparticle of KM01 EPS was found to be reversible. Finally, the suitability of KM01 EPS for biomedical applications was demonstrated by its lack of cytotoxicity and its ability to form complexes with quercetin. Unlike the common α-1,6-linked dextran, KM01 EPS can enhance the solubility of quercetin significantly.

Tetrahydroxanthone-chromanone heterodimers from lichen Usnea aciculifera and their cytotoxic activity against human cancer cell lines.

Four new tetrahydroxanthone-chromanone heterodimers, usneaxanthones E-H (1-4) together with eleven known compounds (5-15) were isolated from lichen Usnea aciculifera Vain (Parmeliaceae). Their structures and absolute configurations, particularly the central and axial chiralities, were unambiguously demonstrated by a combination of spectroscopic data (1D, 2D NMR, HRESIMS), electronic circular dichroism (ECD) experiments, and single-crystal X-ray crystallographic analyses. The cytotoxicity of new compounds was evaluated on four human cancer cell lines including HCT116 colorectal cancer, MCF-7 breast cancer, A549 lung cancer, and OVCAR-3 ovarian cancer. Compounds 1-4 exhibited good cytotoxicity against all tested cancer cell lines, except ovarian cancer, with the best IC50 value of 3.37 µM. All compounds showed potent cytotoxicity against HCT116 colon cancer with IC50 value from 3.37 to 4.53 µM.

Pharmacokinetics of nucleoside/nucleotide reverse transcriptase inhibitors for the treatment and prevention of HIV infection.

INTRODUCTION: Despite dramatic increases in new drugs and regimens, a combination of two nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) remains the backbone of many regimens to treat HIV. AREA COVERED: This article summarizes the pharmacokinetic characteristics of approved NRTIs that are currently in the international treatment and prevention guidelines. EXPERT OPINION: Compared to other NRTIs, tenofovir alafenamide fumarate (TAF) is more advantageous in terms of potency and safety. It is therefore a preferred choice in combination with emtricitabine (FTC) in most HIV treatment guidelines. The efficacy of the two-drug combination of NRTI/Integrase strand-transfer inhibitor, i.e. lamivudine/dolutegravir has been approved as an option for initial therapy. This regimen however has some limitations in patients with HBV coinfection. The two NRTI combinations tenofovir disproxil fumarate (TDF)/FTC and TAF/FTC have also been approved for pre-exposure prophylaxis (PrEP). Interestingly, a promising long-acting nucleoside reverse transcriptase translocation inhibitor, islatravir, formulated for implant was well tolerated and remained effective for up to a year, suggesting its potential as a single agent for PrEP. In the next decade, it remains to be seen whether NRTI-based regimens will remain the backbone of preferred ART regimens, or if the treatment will eventually move toward NRTI-sparing regimens to avoid long-term NRTI-toxicity.

Butoxy Mansonone G Inhibits STAT3 and Akt Signaling Pathways in Non-Small Cell Lung Cancers: Combined Experimental and Theoretical Investigations.

Epidermal growth factor receptor (EGFR) is the key molecular target for non-small cell lung cancer (NSCLC) due to its major contribution to complex signaling cascades modulating the survival of cancer cells. Targeting EGFR-mediated signaling pathways has been proved as a potential strategy for NSCLC treatment. In the present study, mansonone G (MG), a naturally occurring quinone-containing compound, and its semi-synthetic ether derivatives were subjected to investigate the anticancer effects on human NSCLC cell lines expressing wild-type EGFR (A549) and mutant EGFR (H1975). In vitro cytotoxicity screening results demonstrated that butoxy MG (MG3) exhibits the potent cytotoxic effect on both A549 (IC50 of 8.54 µM) and H1975 (IC50 of 4.21 µM) NSCLC cell lines with low toxicity against PCS201-010 normal fibroblast cells (IC50 of 21.16 µM). Western blotting and flow cytometric analyses revealed that MG3 induces a caspase-dependent apoptosis mechanism through: (i) inhibition of p-STAT3 and p-Akt without affecting upstream p-EGFR and (ii) activation of p-Erk. The 500-ns molecular dynamics simulations and the molecular mechanics combined with generalized Born surface area (MM/GBSA)-based binding free energy calculations suggested that MG3 could possibly interact with STAT3 SH2 domain and ATP-binding pocket of Akt. According to principal component analysis, the binding of MG3 toward STAT3 and Akt dramatically altered the conformation of proteins, especially the residues in the active site, stabilizing MG3 mainly through van der Waals interactions.

Ethoxy mansonone G as an anticancer agent in estrogen receptor-positive and endocrine-resistant breast cancer.

OBJECTIVES: To study anticancer effects, underlying mechanism and safety of ethoxy mansonone G (EMG) which is the potent derivative of mansonone G (MG) in breast cancer cells. METHODS: Anticancer, antimigration, anti-invasion effects and anchorage-independent growth were investigated by MTT, scratch, matrigel invasion and soft agar assays. Estrogen receptor (ER)-targeted genes and endocrine-resistant genes were assessed by RT-PCR and Western blot. KEY FINDINGS: Ethoxy mansonone G is the most potent MG derivative and has anticancer effects in ER-positive, endocrine-resistant and ER-negative breast cancer cells. Our results demonstrated that EMG can significantly inhibit estrogen-induced cell proliferation and the expression of ER-targeted genes in ER-positive breast cancer cells, suggesting the anti-estrogenic property of EMG which is consisting with the virtual molecular docking that EMG could possibly bind to the ERα. Moreover, EMG has synergistic effect with tamoxifen in endocrine-resistant cells. EMG also inhibited cell proliferation, invasion and anchorage-independent growth by reducing expression of genes involved in endocrine resistance and invasive factors during the metastatic process. CONCLUSION: Ethoxy mansonone G has an anticancer effect in breast cancer cells and is possible to use as a therapeutic agent in patients with breast cancer.

Enhanced Solubility and Anticancer Potential of Mansonone G By ß-Cyclodextrin-Based Host-Guest Complexation: A Computational and Experimental Study.

Mansonone G (MG), a plant-derived compound isolated from the heartwood of Mansoniagagei, possesses a potent antitumor effect on several kinds of malignancy. However, its poor solubility limits the use for practical applications. Beta-cyclodextrin (ßCD), a cyclic oligosaccharide composed of seven (1→4)-linked α-D-glucopyranose units, is capable of encapsulating a variety of poorly soluble compounds into its hydrophobic interior. In this work, we aimed to enhance the water solubility and the anticancer activity of MG by complexation with ßCD and its derivatives (2,6-di-O-methyl-ßCD (DMßCD) and hydroxypropyl-ßCD). The 90-ns molecular dynamics simulations and MM/GBSA-based binding free energy results suggested that DMßCD was the most preferential host molecule for MG inclusion complexation. The inclusion complex formation between MG and ßCD(s) was confirmed by DSC and SEM techniques. Notably, the MG/ßCDs inclusion complexes exerted significantly higher cytotoxic effect (2-7 fold) on A549 lung cancer cells than the uncomplexed MG.

Dimeric tetrahydroxanthones from the lichen Usnea aciculifera.

Four unusual heterodimeric tetrahydroxanthones, usneaxanthones A-D (1-4) were isolated from lichen Usnea aciculifera Vain (Parmeliaceae). Their structures and absolute configurations, particularly the central and axial chiralities, were unambiguously demonstrated by a combination of spectroscopic data (1D, 2D NMR, HRESIMS), electronic circular dichroism (ECD) experiments, and single-crystal X-ray crystallographic analyses. Cytotoxic effects of isolated compounds (1, 2 and 4) were evaluated on HT-29 human colorectal cancer cells. Compound 4 showed potent cytotoxicity against HT-29 with IC50 values of 2.41 µM.

Molecular and macromolecular alterations of recombinant adenoviral vectors do not resolve changes in hepatic drug metabolism during infection.

In this report we test the hypothesis that long-term virus-induced alterations in CYP occur from changes initiated by the virus that may not be related to the immune response. Enzyme activity, protein expression and mRNA of CYP3A2, a correlate of human CYP3A4, and CYP2C11, responsive to inflammatory mediators, were assessed 0.25, 1, 4, and 14 days after administration of several different recombinant adenoviruses at a dose of 5.7 x 1012 virus particles (vp)/kg to male Sprague Dawley rats. Wild type adenovirus, containing all viral genes, suppressed CYP3A2 and 2C11 activity by 37% and 39%, respectively within six hours. Levels fell to 67% (CYP3A2) and 79% (CYP2C11) of control by 14 days (p

Controlled inactivation of recombinant viruses with vitamin B2.

Inactivated viruses are important tools for vaccine development and gene transfer. 8-Methoxypsoralen (8-MOP) and long-wavelength ultraviolet irradiation (LWUVI) inactivates many viruses. Toxicity limits its use in animals and humans. Toxicological and photosensitizing properties of riboflavin make it suitable for virus inactivation in preparations for biological use. Viruses expressing beta-galactosidase were mixed with either 8-MOP (1.5mM) or riboflavin (50 microM) and exposed to LWUVI (365 nm) for 2 h. Virus activity was determined by limiting dilution. The half-life of the adenovirus preparation treated with 8-MOP was 8.28 ns(-1) and 36.5 ns(-1) after treatment with riboflavin. Despite the difference in half-life, both preparations were completely inactivated within 45 min. In contrast, the half-lives for adeno-associated virus (AAV) preparations were similar (63 ns(-1) 8-MOP vs. 67 ns(-1) riboflavin). Each AAV preparation was fully inactivated within 90 min. The half-life of lentivirus was 193.4 ns(-1) after treatment with 8-MOP and 208 ns(-1) after exposure to riboflavin. Virus treated with riboflavin was inactivated within 20 min. Virus exposed to 8-MOP was inactivated in 90 min. DNA and RNA viruses can be inactivated by riboflavin and LWUVI and used in physiological systems sensitive to other photochemicals.

Cepharanthine exhibits a potent anticancer activity in p53-mutated colorectal cancer cells through upregulation of p21Waf1/Cip1.

Cepharanthine (CEP), a biscoclurine alkaloid isolated from Stephania cepharantha Hayata, has demonstrated anticancer activity in several different types of cancer cells. Colorectal cancer (CRC) is one of the most common cancers in both men and women. Mutated p53 in CRC was reported to be associated with resistance to commonly used chemotherapeutic agents including, 5­fluorouracil, oxaliplatin and irinotecan. Many studies reported that mutation of p53 induced chemoresistance through several mechanisms, including induction of drug efflux, disruption of cell cycle regulation, evasion of apoptosis and upregulation of DNA repair. This study aimed to evaluate the anticancer activity of CEP in p53 mutant versus p53 wild-type colorectal cancer cells and determine its underlying mechanisms of action. Our results showed that CEP induced colorectal cancer cell death in a concentration-dependent manner. Remarkably, CEP was more effective in controlling the growth of the p53 mutant colorectal cancer cell lines, HT­29 and SW-620, than the p53 wild-type colorectal cancer cell lines, COLO­205 and HCT-116. Further studies on the underlying mechanisms revealed that CEP could induce cell cycle arrest and apoptosis in both HT­29 and COLO­205 cells. Treatment with CEP dramatically increased p21Waf1/Cip1 expression levels of the p53 mutant cell line HT­29 and to a lesser extent, the p53 wild-type cell line COLO­205. In addition, cyclin A and Bcl­2 expression levels of both cell lines were significantly downregulated following treatment with CEP. CEP also induced ROS formation in colorectal cancer cells. Taken together, we concluded that CEP effectively induced cell cycle arrest and apoptosis which may be mediated through upregulation of p21Waf1/Cip1, downregulation of cyclin A and Bcl­2 and induction of ROS production in colorectal cancer cells. These findings suggested that CEP could potentially be a novel anticancer agent for p53 mutant colorectal cancer cells which are often resistant to current chemotherapeutic agents.

Just getting into cells is not enough: mechanisms underlying 4-(N)-stearoyl gemcitabine solid lipid nanoparticle's ability to overcome gemcitabine resistance caused by RRM1 overexpression.

Gemcitabine is a deoxycytidine analog that is widely used in the chemotherapy of many solid tumors. However, acquired tumor cell resistance often limits its use. Previously, we discovered that 4-(N)-stearoyl gemcitabine solid lipid nanoparticles (4-(N)-GemC18-SLNs) can overcome multiple acquired gemcitabine resistance mechanisms, including RRM1 overexpression. The present study was designed to elucidate the mechanisms underlying the 4-(N)-GemC18-SLNs' ability to overcome gemcitabine resistance. The 4-(N)-GemC18 in the 4-(N)-GemC18-SLNs entered tumor cells due to clathrin-mediated endocytosis of the 4-(N)-GemC18-SLNs into the lysosomes of the cells, whereas the 4-(N)-GemC18 alone in solution entered cells by diffusion. We substantiated that it is the way the 4-(N)-GemC18-SLNs deliver the 4-(N)-GemC18 into tumor cells that allows the gemcitabine hydrolyzed from the 4-(N)-GemC18 to be more efficiently converted into its active metabolite, gemcitabine triphosphate (dFdCTP), and thus more potent against gemcitabine-resistant tumor cells than 4-(N)-GemC18 or gemcitabine alone. Moreover, we also showed that the RRM1-overexpressing tumor cells were also cross-resistant to cytarabine, another nucleoside analog commonly used in cancer therapy, and 4-(N)-stearoyl cytarabine carried by solid lipid nanoparticles can also overcome the resistance. Therefore, formulating the long-chain fatty acid amide derivatives of nucleoside analogs into solid lipid nanoparticles may represent a platform technology to increase the antitumor activity of the nucleoside analogs and to overcome tumor cell resistance to them.