Mitochondrial Fuel Dependence on Glutamine Drives Chemo-Resistance in the Cancer Stem Cells of Hepatocellular Carcinoma.

Chemo-resistance hinders treatment of patients with hepatocellular carcinoma. Although there are many models that can be found in the literature, the root mechanism to explain chemo-resistance is still not fully understood. To gain a better understanding of this phenomenon, a chemo-resistant line, R-HepG2, was developed from a chemo-sensitive HepG2 line through an exposure of doxorubicin (DOX). The R-HepG2 exhibited a cancer stem cell (CSC) phenotype with an over-expression of P-glycoprotein (P-gp), conferring it a significant enhancement in drug efflux and survival. With these observations, we hypothesize that metabolic alteration in this drug-resistant CSC is the root cause of chemo-resistance. Our results show that, unlike other metabolic-reprogrammed CSCs that exhibit glycolytic phenotype described by the "Warburg effect", the R-HepG2 was metabolically quiescent with glucose independence, high metabolic plasticity, and relied on glutamine metabolism via the mitochondria for its chemo-resistance Intriguingly, drug efflux by P-gp in R-HepG2 depended on the mitochondrial ATP fueled by glutamine instead of glycolytic ATP. Armed with these observations, we blocked the glutamine metabolism in the R-HepG2 and a significant reduction of DOX efflux was obtained. We exploited this metabolic vulnerability using a combination of DOX and metformin in a glutamine-free condition to target the R-HepG2, resulting in a significant DOX sensitization. In conclusion, our findings highlight the metabolic modulation of chemo-resistance in CSCs. We delineate the altered metabolism that drives chemo-resistance and offer a new approach to target this CSC through metabolic interventions.

Extracellular Histones Induced Eryptotic Death in Human Erythrocytes.

BACKGROUND/AIMS: Circulating or extracellular histones (EHs) in the bloodstream act as a damage-associated-molecular-pattern (DAMP) agent that plays a critical role in the pathogenesis of many diseases such as sepsis and sterile inflammation. To date, not much information is available to describe the mechanistic relationship between human erythrocytes and the cytotoxicity of EHs, the protein members from a highly conserved histone family across species. The present study explored this key question with a hypothesis that EHs induce eryptosis. METHODS: Freshly isolated human red blood cells (RBCs) from healthy donors were treated with EHs or agents for positive controls in a physiological buffer for 3 or 24 h. After treatments, flow cytometry was employed to quantify surface phosphatidylserine (PS) exposure from annexin-V-RFP binding, cell shrinkage from flow cytometric forward scatter (FSC) analysis, Ca2+ rise by fluo-4, reactive oxygen species (ROS) production by H2DCFDA, and caspase-3 activation by FAM-DEVD-FMK measurement. Hemolysis and membarne permeabilization were estimated respectively from hemoglobin release into supernatant and calcein leakage from RBC ghosts. RESULTS: With positive controls for validation, EHs in the pathophsyiological range were found to accumulate annexin-V binding on cell surface, decrease FSC, upregulate ROS production, elevate Ca2+ influx and increase caspase-3 activity in a 3-h incubation. Of note, no RBC hemolysis and no calcein release from ghosts were obtained after EHs treatment for 24 h. Interestingly, external Ca2+ was not a prerequisite for the EHs-mediated ROS production and PS externalization. Also, the eryptotic hallmarks in the apoptotic RBCs were partially blocked by heparin and antibody (Ab) against Toll-like receptor 2 (TLR2). CONCLUSION: EHs act as a DAMP agent in the human RBCs that induces eryptosis. The cytotoxic effect is rapid as the hallmarks of eryptosis such as cell shrinkage, surface PS exposure, [Ca2+]i rise, ROS production and caspase-3 activation can be seen 3 h after treatment in a dose-dependent manner. The EHs' cytotoxic effects could be blocked by heparin and the Ab against TLR2.

A comprehensive web tool for toehold switch design.

Motivation: Toehold switches are a class of RNAs with a hairpin loop that can be unfolded upon binding a trigger RNA, thereby exposing a ribosome binding site (RBS) and permitting translation of the reporter protein. They have been shown very useful in detecting a variety of targets including RNAs from Zika and Ebola viruses. The base complementation between the toehold switch and the trigger RNA also makes it sensitive to sequence variations. Design of toehold switches involves a series of considerations related to their sequence properties, structures and specificities. Results: Here we present the first comprehensive web tool for designing toehold switches. We also propose a score for predicting the efficacy of designed toehold switches based on properties learned from ∼180 experimentally tested switches. Availability and implementation: The toehold switch web tool is available at https://yiplab.cse.cuhk.edu.hk/toehold/.

Recent Advances in Surface Plasmon Resonance Imaging Sensors.

The surface plasmon resonance (SPR) sensor is an important tool widely used for studying binding kinetics between biomolecular species. The SPR approach offers unique advantages in light of its real-time and label-free sensing capabilities. Until now, nearly all established SPR instrumentation schemes are based on single- or several-channel configurations. With the emergence of drug screening and investigation of biomolecular interactions on a massive scale these days for finding more effective treatments of diseases, there is a growing demand for the development of high-throughput 2-D SPR sensor arrays based on imaging. The so-called SPR imaging (SPRi) approach has been explored intensively in recent years. This review aims to provide an up-to-date and concise summary of recent advances in SPRi. The specific focuses are on practical instrumentation designs and their respective biosensing applications in relation to molecular sensing, healthcare testing, and environmental screening.

Real-time multi-channel SPR sensing based on DMD-enabled angular interrogation.

This paper reports a digital micro-mirror device (DMD)-enabled real-time multi-channel biosensing system based on angular interrogation surface plasmon resonance (SPR). In the experiments, angular scanning is achieved by a DMD that facilitates SPR measurements using a single-point photodetector. In the four-channel measurement setup, real-time monitoring of bovine serum albumin (BSA) and anti-BSA binding interactions is performed at various concentration levels. The experimental results have verified that the system has a resolution of 3.54 × 10-6 RIU (refractive index unit); and a detection limit of 9 ng/mL. The new DMD-based SPR interrogation system presents a new design route for practical solid-state SPR biosensing with a user-selectable range of interrogation, enhanced signal-to-noise ratio, and fast data throughput.

Development of peptide-based chemiluminescence enzyme immunoassay (CLEIA) for diagnosis of dengue virus infection in human.

Dengue is the most prevalent mosquito-borne viral disease in tropical and subtropical regions worldwide. Since its clinical symptoms are non-specific and easily mistaken as other kinds of infection, laboratory diagnosis is required to confirm dengue infections. In this study, ten peptides (E1-E10) from the envelope protein of dengue virus (DENV) were first identified using bioinformatic tool. The screened peptides were then synthesized for the peptide-based chemiluminescence enzyme immunoassay (CLEIA). Two peptides, E1 and E7, were found as the best candidate antigen and therefore used as downstream application in the development of low-cost peptide-based anti-DENV immunoglobulin M antibodies (IgM) indirect CLEIA. 176 serum samples were used to study the presence of anti-DENV IgM antibodies to evaluate the diagnostic ability of IgM-CLEIA. Receiver operating characteristic curve (ROC) was used to estimate the diagnostic cut-off value. The sensitivity and the specificity reached 82.5% and 94.6% respectively when peptide E1 was used, but declined to 79.2% and 92.9% respectively when peptide E7 was used. Therefore, the combination of E1 and E7 was used to improve the sensitivity and the specificity to 85.0% and 96.4% respectively in 1.5 h assay time, providing a potentially practical use for the diagnosis of DENV infections in patients' serum.

Prevalence and genetic diversity analysis of human coronaviruses among cross-border children.

BACKGROUND: More than a decade after the outbreak of human coronaviruses (HCoVs) SARS in Guangdong province and Hong Kong SAR of China in 2002, there is still no reoccurrence, but the evolution and recombination of the coronaviruses in this region are still unknown. Therefore, surveillance on the prevalence and the virus variation of HCoVs circulation in this region is conducted. METHODS: A total of 3298 nasopharyngeal swabs samples were collected from cross-border children (<6 years, crossing border between Southern China and Hong Kong SAR) showing symptoms of respiratory tract infection, such as fever (body temperature > 37.5 °C), from 2014 May to 2015 Dec. Viral nucleic acids were analyzed and sequenced to study the prevalence and genetic diversity of the four human coronaviruses. The statistical significance of the data was evaluated with Fisher chi-square test. RESULTS: 78 (2.37%; 95%CI 1.8-2.8%) out of 3298 nasopharyngeal swabs specimens were found to be positive for OC43 (36;1.09%), HKU1 (34; 1.03%), NL63 (6; 0.18%) and 229E (2;0.01%). None of SARS or MERS was detected. The HCoVs predominant circulating season was in transition of winter to spring, especially January and February and NL63 detected only in summer and fall. Complex population with an abundant genetic diversity of coronaviruses was circulating and they shared homology with the published strains (99-100%). Besides, phylogenetic evolutionary analysis indicated that OC43 coronaviruses were clustered into three clades (B,D,E), HKU1 clustered into two clades(A,B) and NL63 clustered into two clades(A,B). Moreover, several novel mutations including nucleotides substitution and the insertion of spike of the glycoprotein on the viral surface were discovered. CONCLUSIONS: The detection rate and epidemic trend of coronaviruses were stable and no obvious fluctuations were found. The detected coronaviruses shared a conserved gene sequences in S and RdRp. However, mutants of the epidemic strains were detected, suggesting continuous monitoring of the human coronaviruses is in need among cross-border children, who are more likely to get infected and transmit the viruses across the border easily, in addition to the general public.

An Aptamer Bio-barCode (ABC) assay using SPR, RNase H, and probes with RNA and gold-nanorods for anti-cancer drug screening.

With modifications to an ultra-sensitive bio-barcode (BBC) assay, we have developed a next generation aptamer-based bio-barcode (ABC) assay to detect cytochrome-c (Cyto-c), a cell death marker released from cancer cells, for anti-cancer drug screening. An aptamer is a short single-stranded DNA selected from a synthetic DNA library that is capable of binding to its target with high affinity and specificity based on its unique DNA sequence and 3D structure after folding. Similar to the BBC assay, Cyto-c is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Ab) and an aptamer specifically against Cyto-c to form sandwich structures ([MMP-Ab]-[Cyto-c]-[Aptamer]). After washing and melting, our aptamers, acting as a DNA bio-barcode, are released from the sandwiches and hybridized with the probes specially designed for RNase H for surface plasmon resonance (SPR) sensing. In an aptamer-probe duplex, RNase H digests the RNA in the probe and releases the intact aptamer for another round of hybridization and digestion. With signal enhancement effects from gold-nanorods (Au-NRs) on probes for SPR sensing, the detection limit was found to be 1 nM for the aptamer and 80 pM for Cyto-c. Without the time-consuming DNA amplification steps by PCR, the detection process of this new ABC assay can be completed within three hours. As a proof-of-concept, phenylarsine oxide was found to be a potent agent to kill liver cancer cells with multi-drug resistance at the nano-molar level. This approach thus provides a fast, sensitive and robust tool for anti-cancer drug screening.

Identification of microRNAs in Throat Swab as the Biomarkers for Diagnosis of Influenza.

BACKGROUND: Influenza is a serious worldwide disease that captures global attention in the past few years after outbreaks. The recent discoveries of microRNA (miRNA) and its unique expression profile in influenza patients have offered a new method for early influenza diagnosis. The aim of this study was to examine the utility of miRNAs for the diagnosis of influenza. METHODS: Thirteen selected miRNAs were investigated with the hosts' throat swabs (25 H1N1, 20 H3N2, 20 influenza B and 21 healthy controls) by real-time quantitative polymerase chain reaction (RT-qPCR) using U6 snRNA as endogenous control for normalization, and receiver operating characteristic (ROC) curve/Area under curve (AUC) for analysis. RESULTS: miR-29a-3p, miR-30c-5p, miR-34c-3p and miR-181a-5p are useful biomarkers for influenza A detection; and miR-30c-5p, miR-34b-5p, miR-205-5p and miR-449b-5p for influenza B detection. Also, use of both miR-30c-5p and miR-34c-3p (AUC=0.879); and miR-30c-5p and miR-449b-5p (AUC=0.901) are better than using one miRNA to confirm influenza A and influenza B infection, respectively. CONCLUSIONS: Given its simplicity, non-invasiveness and specificity, we found that the throat swab-derived miRNAs miR-29a-3p, miR-30c-5p, miR-34b-5p, miR-34c-3p, miR-181a-5p, miR-205-5p and miR-449b-5p are a useful tool for influenza diagnosis on influenza A and B.

Plasmonic random nanostructures on fiber tip for trapping live cells and colloidal particles.

We demonstrate optical trapping on a gold-coated single-mode fiber tip as excited by 980-nm laser radiation. The trapping force here is not due to common plasmonic localization, but dominated by the combined effect of thermophoresis and thermal convection. The reported scheme only requires simple thin-film deposition. More importantly, efficient broadband plasmonic absorption of the gold random nanostructures, aided by purely Gaussian excitation profile from the fiber core, has led to very low trapping-power threshold typically in hundreds of microwatts. This highly versatile fiber-based trapping scheme clearly offers many potential application possibilities in life sciences as well as engineering disciplines.

From skeleton to cytoskeleton: osteocalcin transforms vascular fibroblasts to myofibroblasts via angiotensin II and Toll-like receptor 4.

RATIONALE: The expression of osteocalcin is augmented in human atherosclerotic lesions. How osteocalcin triggers vascular pathogenesis and remodeling is unclear. OBJECTIVE: To investigate whether osteocalcin promotes transformation of adventitial fibroblast to myofibroblasts and the molecular mechanism involved. METHODS AND RESULTS: Immunohistochemistry indicated that osteocalcin was expressed in the neointima of renal arteries from diabetic patients. Western blotting and wound-healing assay showed that osteocalcin induced fibroblast transformation and migration, which were attenuated by blockers of the renin-angiotensin system and protein kinase Cδ (PKCδ), toll-like receptor 4 (TLR4) neutralizing antibody, and antagonist and inhibitors of free radical production and cyclooxygenase-2. Small interfering RNA silencing of TLR4 and PKCδ abolished fibroblast transformation. Angiotensin II level in the conditioned medium from the osteocalcin-treated fibroblasts was found elevated using enzyme immunoassay. Culturing of fibroblasts in conditioned medium collected from differentiated osteoblasts promoted fibroblast transformation. The expression of fibronectin, TLR4, and cyclooxygenase-2 is augmented in human mesenteric arteries after 5-day in vitro exposure to osteocalcin. CONCLUSIONS: Osteocalcin transforms adventitial fibroblasts to myofibroblasts through stimulating angiotensin II release and subsequent activation of PKCδ/TLR4/reactive oxygen species/cyclooxygenase-2 signaling cascade. This study reveals that the skeletal hormone osteocalcin cross-talks with vascular system and contributes to vascular remodeling.

Common-path spectral interferometry with temporal carrier for highly sensitive surface plasmon resonance sensing.

Incorporating the temporal carrier technique with common-path spectral interferometry, we have successfully demonstrated an advanced surface plasmon resonance (SPR) biosensing system which achieves refractive index resolution (RIR) up to 2 × 10(-8) refractive index unit (RIU) over a wide dynamic range of 3 × 10(-2) RIU. While this is accomplished by optimizing the SPR differential phase sensing conditions with just a layer of gold, we managed to address the spectral phase discontinuity with a novel spectral-temporal phase measurement scheme. As the new optical setup supersedes its Michelson counterpart in term of simplicity, we believe that it is a significant contribution for practical SPR sensing applications.

Ferutinin induces in vitro eryptosis/erythroptosis in human erythrocytes through membrane permeabilization and calcium influx.

Ferutinin, isolated from the root of Ferula hermonis and proposed to be used as an antiosteoporosis phytoestrogen, has death promoting activities in a number of cancer cells. However, the effect of ferutinin on the induction of apoptosis in human red blood cells (RBCs), also known as eryptosis or erythroptosis, remains unclear. Given that ferutinin is a small molecule that can induce apoptosis in the cancer cells by opening the mitochondrial permeability transition pores, we therefore hypothesized that the effect of ferutinin to elicit apoptosis in human RBCs devoid of mitochondria would be minimal. This study tried to determine the in vitro effect of ferutinin on the induction of apoptosis in human RBCs. Eryptosis/erythroptosis after ferutinin treatment was examined for phosphatidylserine (PS) externalization, calcein leakage, and other apoptotic feature events by flow cytometry and confocal microscopy. Contrary to our prediction, ferutinin caused eryptosis/erythroptosis in human RBCs and simultaneously increased caspase-3 activity and the cytosolic free Ca(2+) ion level ([Ca(2+)]i). Yet, Ca(2+) seems not to be the sole mediator in ferutinin-mediated eryptosis/erythroptosis because depletion of the external Ca(2+) could not eliminate the apoptotic effect from ferutinin. Subsequent replenishment of the external Ca(2+) was able to promote PS externalization, caspase-3 activation, and rise of [Ca(2+)]i. Also, ferutinin at high dose (40 µM or above) was able to permeabilize the membrane of RBC ghosts in a way similar to that of digitonin. At low dose, ferutinin activated the P- and L-type Ca(2+) channels as the ferutinin-mediated [Ca(2+)]i rise was suppressed by the P-type (ω-agatoxin IVA) and L-type (verapamil and diltiazem) Ca(2+) channel blockers. Taken together, we report here for the first time that ferutinin induces in vitro apoptosis in human RBCs. Mechanistically, eryptosis/erythroptosis is mediated by membrane permeabilization and upregulation of [Ca(2+)]i with the activation of caspase-3.

Bone morphogenic protein-4 induces endothelial cell apoptosis through oxidative stress-dependent p38MAPK and JNK pathway.

The expression of bone morphogenic protein 4 (BMP4), a new pro-inflammatory marker, is increased by disturbed flow in endothelial cells (ECs). BMP4 stimulates production of reactive oxygen species (ROS) and causes endothelial cell dysfunction. The present study examined BMP4-induced apoptosis in ECs and isolated arteries from rat, mouse, and human, and the signaling pathways mediating BMP4-induced apoptosis. Apoptosis was assessed by flow cytometry to detect Annexin-V positive cells, and terminal deoxynucleotidyl transferase dUTP nick end (TUNEL) labeling. The superoxide production was measured by dihydroethidium fluorescence. BMP4 induced EC apoptosis in human mesenteric arteries, mouse aortic endothelium, rat primary ECs, and human ECs. BMP4-induced EC apoptosis was mediated through ROS production by activation of NADPH oxidase, which led to cleaved caspase-3 expression. BMP4 also induced sequential activation of p38 MAPK and JNK which was upstream of caspase 3 activation. Knockdown of BMP receptor 1A by lentiviral shRNA or NOX4 siRNA transfection inhibited BMP4-induced ROS production, p38 and JNK phosphorylation, and caspase-3 activation in ECs. JNK siRNA inhibited BMP4-induced JNK phosphorylation and caspase-3 activation. The present study delineates that BMP4 causes EC apoptosis through activation of caspase-3 in a ROS/p38MAPK/JNK-dependent signaling cascade.

Salidroside protects human erythrocytes against hydrogen peroxide-induced apoptosis.

Rhodiola rosea is a commonly used folk medicine for the treatment of high altitude sickness, mountain malhypoxia, and anoxia. Its active ingredient, salidroside [2-(4-hydroxyphenyl)ethyl ß-D-glucopyranoside (1)], has been reported to have a broad spectrum of biological effects. However, the protective role of 1 in human erythrocytes remains unclear. This study therefore has investigated the effects of 1 on oxidative stress-induced apoptosis in human erythrocytes (also known as eryptosis or erythroptosis). Compound 1 increased cell survival significantly and prevented human erythrocytes from undergoing eryptosis/erythroptosis mediated by H(2)O(2), as confirmed by the decreased expression of phosphatidylserine on the cell surface and reduced leakage of calcein through the damaged membrane. Mechanistically, 1 was found to exert its protective effects through its antioxidative activity and the inhibition of caspase-3 activation and stress-induced intracellular Ca(2+) rise in a dose-dependent manner. Compound 1 is a protective agent in human erythrocytes against oxidative stress and may be a good adaptogen to enhance the body's resistance to stress and fatigue.

Stem cell differentiation with consistent lineage commitment induced by a flash of ultrafast-laser activation in vitro and in vivo.

Recent technological advancements on stem cell differentiation induction have been making great progress in stem cell research, regenerative medicine, and therapeutic applications. However, the risk of off-target differentiation limits the wide application of stem cell therapy strategies. Here, we report a non-invasive all-optical strategy to induce stem cell differentiation in vitro and in vivo that activates individual target stem cells in situ by delivering a transient 100-ms irradiation of a tightly focused femtosecond laser to a submicron cytoplasmic region of primary adipose-derived stem cells (ADSCs). The ADSCs differentiate to osteoblasts with stable lineage commitment that cannot further transdifferentiate because of simultaneous initiation of multiple signaling pathways through specific Ca2+ kinetic patterns. This method can work in vivo to direct mouse cerebellar granule neuron progenitors to granule neurons in intact mouse cerebellums through the skull. Hence, this optical method without any genetic manipulations or exogenous biomaterials holds promising potential in biomedical research and cell-based therapies.

A high spatial resolution osteogenic differentiation in human mesenchymal stem cells induced by femtosecond laser.

A variety of physical and chemical methods have been developed in research laboratories for the induction of stem cell differentiation. However, the use of exogenous chemicals and materials may limit their widespread utility in clinics. To develop a clean and precise induction approach with minimal invasion, we reported here that 1-second stimulation by a tightly focused femtosecond laser (fsL) (140 mW/µm2 , 200 fs) can modulate the signaling systems in human mesenchymal cells, such as intracellular calcium and reactive oxygen species. Upon stimulation on an automatic platform, hMSCs were found to express osteoblastic markers and form calcium-rich deposits. Moreover, tissue mineralization was observed when the fsL-illuminated hMSCs were ectopically transplanted into nude mice. Collectively, we described a novel and non-contact optical stimulation method for cell differentiation with high spatiotemporal resolution.

P-glycoprotein enhances radiation-induced apoptotic cell death through the regulation of miR-16 and Bcl-2 expressions in hepatocellular carcinoma cells.

P-glycoprotein (Pgp), an efflux pump, was confirmed the first time to regulate the expressions of miR/gene in cells. Pgp is known to be associated with multidrug resistance. RHepG2 cells, the multidrug resistant subline of human hepatocellular carcinoma HepG2 cells, expressed higher levels of Pgp as well as miR-16, and lower level of Bcl-2 than the parental cells. In addition, RHepG2 cells were more radiation sensitive and showed more pronounced radiation-induced apoptotic cell death than the parental cells. Mechanistic analysis revealed that transfection with mdr1 specific antisense oligos suppressed radiation-induced apoptosis in HepG2 cells. On the other hand, ectopic mdr1 expression enhanced radiation-induced apoptosis in HepG2 cells, SK-HEP-1 cells, MiHa cells, and furthermore, induced miR-16 and suppressed its target gene Bcl-2 in HepG2 cells. Moreover, the enhancement effects of Pgp and miR-16 on radiation-induced apoptosis were counteracted by overexpression of Bcl-2. The Pgp effect on miR-16/Bcl-2 was suppressed by Pgp blocker verapamil indicating the importance of the efflux of Pgp substrates. The present study is the first to reveal the role of Pgp in regulation of miRNA/gene expressions. The findings may provide new perspective in understanding the biological function of Pgp.

Antibody-free rapid diagnosis of malaria in whole blood with surface-enhanced Raman Spectroscopy using Nanostructured Gold Substrate.

PURPOSE: The aim of this study is to establish a rapid antibody-free diagnostic method of malaria infection with Plasmodium falciparum and Plasmodium vivax in whole blood with Surface-enhanced Raman Spectroscopy using Nanostructured Gold Substrate. MATERIALS AND METHODS: The blood samples collected from patients were first lysed and centrifuged before dropping on the gold nano-structure (AuNS) substrate. Malaria diagnosis was performed by detecting Raman peaks from Surface Enhanced Raman Spectroscopy (SERS) with a 532 nm laser excitation. RESULTS: Raman peaks at 1370 cm-1, 1570 cm-1, and 1627 cm-1, known to have high specificity against interference from other mosquito-borne diseases such as Dengue and West Nile virus infection, were selected as the fingerprint markers associated with P. falciparum and P. vivax infection. The limit of detection was 10-5 dilution, corresponding to the concentration of parasitized blood cells of 100/mL. A total number of 25 clinical samples, including 5 from patients with P. falciparum infection, 10 with P. vivax infection and 10 from healthy volunteers, were evaluated to support its clinical practical use. The whole assay on malaria detection took 30 min to complete. CONCLUSIONS: While the samples analyzed in this work have strong clinical relevance, we have clearly demonstrated that sensitive malaria detection using AuNS-SERS is a practical direction for rapid in-field diagnosis of malaria infection.

Photodynamic therapy inhibits P-glycoprotein mediated multidrug resistance via JNK activation in human hepatocellular carcinoma using the photosensitizer pheophorbide a.

BACKGROUND: Multidrug resistance (MDR) is frequently observed after prolonged treatment in human hepatoma with conventional anti-tumor drugs, and photodynamic therapy (PDT) is a recently suggested alternative to overcome MDR. The therapeutic potential of PDT was evaluated in a multidrug resistance (MDR) human hepatoma cell line R-HepG2 with photosensitizer pheophorbide a (Pa). RESULTS: Our results demonstrated that intracellular accumulation of Pa was not reduced by the overexpression of P-glycoprotein. Pa-based PDT (Pa-PDT) significantly inhibited the growth of R-HepG2 cells with an IC50 value of 0.6 microM. Mechanistic study demonstrated that genomic DNA fragmentation and phosphatidylserine externalization occurred where increase of intracellular singlet oxygen level triggers the phosphorylation of c-Jun N-terminal Kinase (JNK) and leads to activation of intrinsic apoptotic caspases cascade during the Pa-PDT treatment. The cytotoxicity of Pa-PDT, accumulation of sub-G1 population, and depolarization of mitochondrial membrane could be inhibited by JNK inhibitor in the Pa-PDT treated cells. Interestingly, the Pa-PDT induced JNK activation showed inhibitory effect on MDR by the down-regulation of P-glycoprotein in R-HepG2 cells in a dose-dependent manner. In addition, significant reduction of tumor size was obtained in Pa-PDT treated R-HepG2-bearing nude mice with no significant damages in liver and heart. CONCLUSION: In summary, our findings provided the first evidence that PDT could inhibit the MDR activity by down-regulating the expression of P-glycoprotein via JNK activation using pheophorbide a as the photosensitizer, and our work proved that Pa-PDT inhibited the growth of MDR hepatoma cells by mitochondrial-mediated apoptosis induction.