Cell wall reconstruction and DNA damage repair play a key role in the improved salt tolerance effects of He-Ne laser irradiation in tall fescue seedlings

The improved salt tolerance effects of He–Ne laser were further studied through the estimation of ROS levels, cell viability, DNA damage phenomena, physicochemical properties, and monosaccharide compositions of cell wall polysaccharides in tall fescue seedlings. Salt stress produced deleterious effects on seedlings growth and development. ROS levels and genomic DNA damage were markedly increased compared with controls. Physicochemical activities and monosaccharide proportions of cell wall polysaccharide were also pronouncedly altered. He–Ne laser irradiation improved plant growth retardation via increasing cell viability and reverting physicochemical parameters. According to the results of Fourier transform infrared (FTIR) scanning spectra and DNA apopladder analysis, He–Ne laser was showed to efficiently ameliorate cell wall polysaccharide damage and DNA fragmentation phenomena. The treatment with DNA synthesis inhibitor further demonstrated that DNA damage repair was correlated with the improvement effects of the laser. Therefore, our data illustrated that He–Ne laser irradiation resulted in cell wall reconstruction and genomic DNA injury repair in vivo in salt-stressed seedlings, then enhanced salt tolerance probably via interactions between plant cell wall and related resistance gene expression pattern.     

Label‐free differentiation of human immunodeficiency virus‐1 infected from uninfected cells using transmission measurement

Transmission measurement has been perceived as a potential candidate for label‐free investigation of biological material. It is a real‐time, label‐free and non‐invasive optical detection technique that has found wide applications in pharmaceutical industry as well as the biological and medical fields. Combining transmission measurement with optical trapping has emerged as a powerful tool allowing stable sample trapping, while also facilitating transmittance data analysis. In this study, a near‐infrared laser beam emitting at a wavelength of 1064 nm was used for both optical trapping and transmission measurement investigation of human immunodeficiency virus 1 (HIV‐1) infected and uninfected TZM‐bl cells. The measurements of the transmittance intensity of individual cells in solution were carried out using a home built optical trapping system combined with laser transmission setup using a single beam gradient trap. Transmittance spectral intensity patterns revealed significant differences between the HIV‐1 infected and uninfected cells. This result suggests that the transmittance data analysis technique used in this study has the potential to differentiate between infected and uninfected TZM‐bl cells without the use of labels. The results obtained in this study could pave a way into developing an HIV‐1 label‐free diagnostic tool with possible applications at the point of care .     

The involvement of human RECQL4 in DNA double‐strand break repair

Rothmund–Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas. The precise role of RECQL4 in cellular pathways is largely unknown; however, recent evidence suggests its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double‐strand break (DSB) repair. The results show that RECQL4‐deficient fibroblasts are moderately sensitive to γ‐irradiation and accumulate more γH2AX and 53BP1 foci than control fibroblasts. This is suggestive of defects in efficient repair of DSB’s in the RECQL4‐deficient fibroblasts. Real time imaging of live cells using laser confocal microscopy shows that RECQL4 is recruited early to laser‐induced DSBs and remains for a shorter duration than WRN and BLM, indicating its distinct role in repair of DSBs. Endogenous RECQL4 also colocalizes with γH2AX at the site of DSBs. The RECQL4 domain responsible for its DNA damage localization has been mapped to the unique N‐terminus domain between amino acids 363–492, which shares no homology to recruitment domains of WRN and BLM to the DSBs. Further, the recruitment of RECQL4 to laser‐induced DNA damage is independent of functional WRN, BLM or ATM proteins. These results suggest distinct cellular dynamics for RECQL4 protein at the site of laser‐induced DSB and that it might play important roles in efficient repair of DSB’s.     

Role of caffeine in DNA recognition of a potential food‐carcinogen benzo[a]pyrene and UVA induced DNA damage

Electron transfer (ET) reactions are important for their implications in both oxidative and reductive DNA damages. The current contribution investigates the efficacy of caffeine, a xanthine alkaloid in preventing UVA radiation induced ET from a carcinogen, benzo[a]pyrene (BP) to DNA by forming stable caffeine–BP complexes. While steady‐state emission and absorption results emphasize the role of caffeine in hosting BP in aqueous medium, the molecular modeling studies propose the energetically favorable structure of caffeine–BP complex. The picosecond‐resolved emission spectroscopic studies precisely explore the caffeine‐mediated inhibition of ET from BP to DNA under UVA radiation. The potential therapeutic activity of caffeine in preventing DNA damage has been ensured by agarose gel electrophoresis. Furthermore, time‐gated fluorescence microscopy has been used to monitor caffeine‐mediated exclusion of BP from various cell lines including squamous epithelial cells, WI‐38 (fibroblast), MCF‐7 (breast cancer) and HeLa (cervical cancer) cells. Our in vitro and ex vivo experimental results provide imperative evidences about the role of caffeine in modified biomolecular recognition of a model carcinogen BP by DNA resulting dissociation of the carcinogen from various cell lines, implicating its potential medicinal applications in the prevention of other toxic organic molecule induced cellular damages. Copyright © 2014 John Wiley & Sons, Ltd.     

DNA‐binding affinity and nuclease activity of two cytotoxic copper terpyridine complexes

Two copper(II) terpyridine complexes, [Cu(atpy)(NO₃)(H₂O)](NO₃) ? 3H₂O ( 1 ) and [Cu(ttpy)(NO₃)₂] ( 2 ) (atpy = 4′‐p‐N9‐adeninylmethyl‐phenyl‐2,2′:6,2″‐terpyridine; ttpy = 4′‐p‐tolyl‐2,2′:6,2″‐terpyridine) exhibited high cytotoxicity, with average ten times more potency than cisplatin against the human cervix carcinoma cell line (HeLa), the human liver carcinoma cell line (HepG2), the human galactophore carcinoma cell line (MCF7), and the human prostate carcinoma cell line (PC‐3). The cytotoxicity of the complex 1 was lower than that of the complex 2 . Both complexes showed more efficient oxidative DNA cleavage activity under irradiation with UV light at 260 nm than in the presence of ascorbic acid. Especially, complex 1 exhibited evident photoinduced double‐stranded DNA cleavage activity. The preliminary mechanism experiments revealed that hydrogen peroxide was involved in the oxidative DNA damage induced by both complexes. From the absorption titration data, the DNA‐binding affinity of the complexes with surpersoiled plasmid pUC19 DNA, polydAdT, and polydGdC was calculated and complex 2 showed higher binding affinity than complex 1 with all these substrates. The DNA cleavage ability and DNA‐binding affinity of both complexes depended on the substituent group on the terpyrdine ligands. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:295–302, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20292     

Protein kinase Cδ and caspase‐3 modulate TRAIL‐induced apoptosis in breast tumor cells

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL‐induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL‐induced apoptosis was significantly inhibited in PKCδ overexpressing MCF‐7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase‐8 activation, a first step in TRAIL‐induced apoptosis, thus preventing TRAIL‐induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase‐8 activation leading to TRAIL‐induced apoptosis. To determine if caspase‐3‐induced PKCδ cleavage reverses its inhibition on caspase‐8, we developed stable cell lines that either expresses wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) or caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδ mut) utilizing MCF‐7 cells expressing caspase‐3. Cells that overexpress caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδmut) significantly inhibited TRAIL‐induced apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. In MCF‐7/cas‐3/PKCδmut cells, TRAIL‐induced caspase‐8 activation was blocked leading to inhibition of apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase‐8 activation leading to inhibition of TRAIL‐induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase‐3 sensitizes cells to TRAIL‐induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy. J. Cell. Biochem. 111: 979–987, 2010. © 2010 Wiley‐Liss, Inc.     

High fluence low‐power laser irradiation induces mitochondrial permeability transition mediated by reactive oxygen species

High fluence low‐power laser irradiation (HF‐LPLI) can induce cell apoptosis via the mitochondria/caspase‐3 pathway. Here, we further investigated the mechanism involved in the apoptotic process in human lung adenocarcinoma cells (ASTC‐a‐1) at a laser irradiation fluence of 120 J/cm² (633 nm). Cytochrome c release was ascribed to mitochondrial permeability transition (MPT) because the release was prevented by cyclosporine (CsA), a specific inhibitor of MPT. Furthermore, mitochondrial permeability for calcein (～620 Da) was another evidence for the MPT induction under HF‐LPLI treatment. A high‐level intracellular reactive oxygen species (ROS) generation was observed after irradiation. The photodynamically produced ROS caused onset of MPT, as the ROS scavenger docosahexaenoic acid (DHA) prevented the MPT. However, CsA failed to prevented cell death induced by HF‐LPLI, indicating the existence of other signaling pathways. Following laser irradiation, Bax activation occurred after mitochondrial depolarization and cytochrome c release, indicating Bax activation was a downstream event. In the presence of CsA, Bax was still activated at the end‐stage of apoptotic process caused by HF‐LPLI, suggesting that Bax was involved in an alternative‐signaling pathway, which was independent of MPT. Under HF‐LPLI treatment, cell viabilities due to pre‐treatment with DHA, CsA, or Bax small interfering RNA (siRNA) demonstrated that the MPT signaling pathway was dominant, while Bax signaling pathway was secondary, and more importantly ROS mediated both pathways. Taken together, these results showed that HF‐LPLI induced cell apoptosis via the CsA‐sensitive MPT, which was ROS‐dependent. Furthermore, there existed a secondary signaling pathway through Bax activation. The observed link between MPT and triggering ROS could be a fundamental phenomenon in HF‐LPLI‐induced cell apoptosis. J. Cell. Physiol. 218: 603–611, 2009. © 2008 Wiley‐Liss, Inc.     

Comet assay measurements of DNA damage in cells by laser microbeams and trapping beams with wavelengths spanning a range of 308 nm to 1064 nm

DNA damage induced in NC37 lymphoblasts by optical tweezers with a continuous-wave Ti:sapphire laser and a continuous-wave Nd:YAG laser (60-240 mW; 10-50 TJ/m2; 30-120 s irradiation) was studied with the comet assay, a single-cell technique used to detect DNA fragmentation in genomes. Over the wavelength range of 750-1064 nm, the amount of damage in DNA peaks at around 760 nm, with the fraction of DNA damage within the range of 750-780 nm being a factor of two larger than the fraction of DNA damage within the range of 800-1064 nm. The variation in DNA damage was not significant over the range of 800-1064 nm. When the logarithm of damage thresholds measured in the present work, as well as values reported previously in the UV range, was plotted as a function of wavelength, a dramatic wavelength dependence became apparent. The damage threshold values can be fitted on two straight lines, one for continuous-wave sources and the other for pulsed sources, irrespective of the type of source used (e.g. classical lamp or laser). The damage threshold around 760 nm falls on the line extrapolated from values for UV-radiation-induced damage, while the data for 800-1064 nm fall on a line that has a different slope. The change in the slope between 320 and 340 nm observed earlier is consistent with a well-known change in DNA-damaging mechanisms. The change observed around 780 nm is therefore suggestive of a further change in the mechanism(s). The data from this work together with our previous measurements provide, to the best of our knowledge, the most comprehensive view available of the DNA damage produced by microfocused light.     

Synthesis of 3‐Methylidene‐1‐tosyl‐2,3‐dihydroquinolin‐4(1H)‐ones as Potent Cytotoxic Agents

An efficient synthetic strategy to 3‐methylidene‐2,3‐dihydroquinolin‐4(1H)‐ones variously substituted in position 2 has been developed. The title compounds were synthesized in the reaction sequence involving reaction of diethyl methylphosphonate with methyl 2‐(tosylamino)benzoate, condensation of thus formed diethyl 2‐oxo‐2‐(2‐N‐tosylphenyl)ethylphosphonate with various aldehydes followed by successful application of the obtained 3‐(diethoxyphosphoryl)‐1,2‐dihydroquinolin‐4‐ols as Horner–Wadsworth–Emmons reagents for the olefination of formaldehyde. Also, enantioselective approach to the target compounds has been evaluated using 3‐dimenthoxyphosphoryl group as a chiral auxiliary. Single X‐ray crystal analysis of (2S)‐3‐(dimenthoxyphosphoryl)‐2‐phenyl‐1‐tosyldihydroquinolin‐4‐ol revealed the presence of strong resonance‐assisted hydrogen bond (RAHB). The obtained 3‐methylidene‐2,3‐dihydroquinolin‐4(1H)‐ones were then tested for their cytotoxic activity against two leukemia cell lines NALM‐6 and HL‐60 and a breast cancer MCF‐7 cell line. All compounds showed very high cytotoxic activity with the IC₅₀ values mostly below 1 μm in all three cancer cell lines. The selected analogs were also tested on human umbilical vein endothelial cells (HUVEC) and on human mammary gland/breast cells (MCF‐10A) to evaluate their influence on normal cells. Since one of the most serious problems in cancer chemotherapy is the development of drug resistance, the mRNA levels and activity of ABCB1 transporter considered to be the most important factor engaged in drug resistance, were evaluated in MCF‐7 cells treated with two selected analogs. Both compounds were strong ABCB1 transporter inhibitors that could prevent efflux of anticancer drugs from cancer cells.     

Nitric oxide‐mediated inhibition of NFκB regulates hyperthermia‐induced apoptosis

Ascertaining the upstream regulatory mechanisms of hyperthermia‐induced apoptosis is important to understand the role of hyperthermia in combined modality cancer therapy. Accordingly, we investigated whether (i) hyperthermia‐induced apoptosis is mediated through the nitric oxide (NO) signaling pathway and (ii) inhibition of post‐translational modification of IκBα and down regulation of NFκB‐DNA binding activity is an intermediate step in NO‐dependent apoptosis in MCF‐7 breast cancer cells. For hyperthermia treatment, the cells were exposed to 43°C. Intracellular NO levels measured by the fluorescent intensity of DAF‐2A and iNOS expression by immunobloting revealed an increased level of iNOS dependent NO production after 43°C. Apoptosis measured by Annexin V expression and cell survival by clonogenic assay showed a 20% increase in apoptosis after 43°C treatments. EMSA analysis showed a dose‐dependent inhibition of NFκB‐DNA binding activity. The hyperthermia‐mediated inhibition of NFκB was persistent even after 48 h. Inhibition of NO by L ‐NAME rescued the NFκB‐DNA binding activity and inhibits heat‐induced apoptosis. Similarly, over‐expression of NFκB by transient transfection inhibits heat‐induced apoptosis. These results demonstrate that apoptosis upon hyperthermia exposure of MCF‐7 cells is regulated by NO‐mediated suppression of NFκB. J. Cell. Biochem. 106: 999–1009, 2009. © 2009 Wiley‐Liss, Inc.     

Osteoprotegerin production by breast cancer cells is suppressed by dexamethasone and confers resistance against TRAIL‐induced apoptosis

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF‐κB ligand (RANKL) and TNF‐related apoptosis‐inducing ligand (TRAIL). While RANKL is essential for osteoclastogenesis and facilitates breast cancer migration into bone, TRAIL promotes breast cancer apoptosis. We analyzed the expression of OPG and TRAIL and its modulation in estrogen receptor‐positive MCF‐7 cells and receptor‐negative MDA‐MB‐231 cells. In both cells, OPG mRNA levels and protein secretion were dose‐ and time‐dependently enhanced by interleukin (IL)‐1β and suppressed by dexamethasone. In contrast to MCF‐7 cells, MDA‐MB‐231 abundantly expressed TRAIL mRNA, which was enhanced by IL‐1β and inhibited by dexamethasone. TRAIL activated pro‐apoptotic caspase‐3, ‐7, and poly‐ADP‐ribose polymerase and decreased cell numbers of MDA‐MB‐231, but had no effect on MCF‐7 cells. Gene silencing siRNA directed against OPG resulted in a 31% higher apoptotic rate compared to non‐target siRNA‐treated MDA‐MB‐231 cells. Furthermore, TRAIL induced significantly less apoptosis in cells cultured in conditioned media (containing OPG) compared to cells exposed to TRAIL in fresh medium lacking OPG (P < 0.01) and these protective effects were reversed by blocking OPG with its specific ligand RANKL (P < 0.05). The association between cancer cell survival and OPG production by MDA‐MB‐231 cells was further supported by the finding, that modulation of OPG secretion using IL‐1β or dexamethasone prior to TRAIL exposure resulted in decreased and increased rate of apoptosis, respectively (P < 0.05). Thus, OPG secretion by breast cancer cells is modulated by cytokines and dexamethasone, and may represent a critical resistance mechanism that protects against TRAIL‐induced apoptosis. J. Cell. Biochem. 108: 106–116, 2009. © 2009 Wiley‐Liss, Inc.     

Lead‐induced DNA damage and cell apoptosis in human renal proximal tubular epithelial cell: Attenuation via N‐acetyl cysteine and tannic acid

This study investigates the exposure of lead‐induced reactive oxygen species (ROS) generation, DNA damage, and apoptosis and also evaluates the therapeutic intervention using antioxidants in human renal proximal tubular cells (HK‐2 cells). Following treatment of HK‐2 cells with an increasing concentration of lead nitrate (0–50 μM) for 24 h, the intracellular ROS level increased whereas the GSH level decreased significantly in a dose‐dependent manner. Comet assay results revealed that lead nitrate showed the ability to increase the levels of DNA strand breaks in HK‐2 cells. Lead exposure also induced apoptosis through caspase‐3 activation at 30 μg/mL. Pretreatment with N‐acetylcysteine (NAC) and tannic acid showed a significant ameliorating effect on lead‐induced ROS, DNA damage, and apoptosis. In conclusion, lead induces ROS, which may exacerbate the DNA damage and apoptosis via caspase‐3 activation. Additionally, supplementation of antioxidants such as NAC and tannic acid may be used as salvage therapy for lead‐induced DNA damage and apoptosis in an exposed person.     

用积分球系统测量大鼠组织在不同光阑孔径下对氦-氖激光的温反射特性

本文对大鼠组织在不同光阑孔径下对He-Ne激光的漫反射特性进行了测量。实验采用了不同光阑孔径的积分球系统及He-Ne激光,并根据测量数据计算和分析了大鼠组织对该波长激光的漫反射特性。结果表明,有些大鼠组织在632.8nm波长的He-Ne激光照射下对不同的光阑孔径其漫反射率没有显著性区别,而有些大鼠组织在632.8nm波长的He-Ne激光照射下对不同的光阑孔径其漫长反射率有显著性区别,提示在测定不同生物组织的光学特性时,应考虑组织样品的受光照射的面积的大小对测定结果的精确度的影响。     

Physiological monitoring of optically trapped cells: assessing the effects of confinement by 1064-nm laser tweezers using microfluorometry.

We report the results of microfluorometric measurements of physiological changes in optically trapped immotile Chinese hamster ovary cells (CHOs) and motile human sperm cells under continuous-wave (CW) and pulsed-mode trapping conditions at 1064 nm. The fluorescence spectra derived from the exogenous fluorescent probes laurdan, acridine orange, propidium iodide, and Snarf are used to assess the effects of optical confinement with respect to temperature, DNA structure, cell viability, and intracellular pH, respectively. In the latter three cases, fluorescence is excited via a two-photon process, using a CW laser trap as the fluorescence excitation source. An average temperature increase of < 0.1 +/- 0.30 degrees C/100 mW is measured for cells when held stationary with CW optical tweezers at powers of up to 400 mW. The same trapping conditions do not appear to alter DNA structure or cellular pH. In contrast, a pulsed 1064-nm laser trap (100-ns pulses at 40 microJ/pulse and average power of 40 mW) produced significant fluorescence spectral alterations in acridine orange, perhaps because of thermally induced DNA structural changes or laser-induced multiphoton processes. The techniques and results presented herein demonstrate the ability to perform in situ monitoring of cellular physiology during CW and pulsed laser trapping, and should prove useful in studying mechanisms by which optical tweezers and microbeams perturb metabolic function and cellular viability.     

In‐vitro investigations on laser‐induced smoke generation mimicking the laparoscopic laser surgery purposes

Intraoperative smoke‐generation limits the quality of vision during laparoscopic/endoscopic laser‐assisted surgeries. The current study aimed at the evaluation of factors affecting this phenomenon. As a first step, a suitable experimental setup and a test tissue model were established for this investigation. The experimental setup is composed of a specific sample container, a laser therapy component suitable for the ablation of model tissue at different treatment wavelengths (λ = 980 nm, 1350 nm, 1470 nm), a suction unit providing continuous smoke extraction, and a detection unit for smoke quantification via detection of light (λ = 633 nm) scattered from smoke particles. The ablation rate (AR) was calculated by dividing the ablated volume by the ablation time (60 sec). The laser‐induced scattering signal intensity of the smoke (SI) was determined from time‐charts of the signal intensity as a measure for vision, in addition a delay‐time t_delay could be derived defining the onset of SI after the laser was switched on. The ratio SI/AR is used as a measure for smoke generation in relation to the ablation rate. Additionally the light transmission of the tissue samples was used to estimate their optical properties. In this set‐up, smoke generation using λ = 980 nm as ablation laser wavelength was detected after a delay‐time t_delay = (121.6 ± 24.8) sec which is significantly longer compared to the wavelengths λ = 1350 nm with t_delay = (89.8 ± 19.3) sec and λ = 1470 nm with t_delay = (24.7 ± 5.4) sec. Thus, the delay

Experimental set‐up consisting of sample container, laser therapy component, suction unit and scattered‐light detection compartment. time is wavelength‐dependent. The SI/AR ratio was significantly different (p < 0.001) for 1470 nm irradiation compared to 980 nm irradiation [SI/AR(1470) = (11.8 ± 2.6) · 10³ vs. SI/AR(980) = (8.6 ± 2.0) · 10³]. The ablation crater for 980 nm irradiation was comparable with 1470 nm irradiation, but the coagulation rim was thicker in the 980 nm case. In conclusion, it could be shown experimentally that smoke‐generation depends on the wavelength used for laser ablation.     

Imaging of the DNA damage‐induced dynamics of nuclear proteins via nonlinear photoperturbation

Understanding the cellular response to DNA strand breaks is crucial to decipher the mechanisms maintaining the integrity of our genome. We present a novel method to visualize how the mobility of nuclear proteins changes in response to localized DNA damage. DNA strand breaks are induced via nonlinear excitation with femtosecond laser pulses at λ = 1050 nm in a 3D‐confined subnuclear volume. After a time delay of choice, protein mobility within this volume is analysed by two‐photon photoactivation of PA‐GFP fusion proteins at λ = 775 nm. By changing the position of the photoactivation spot with respect to the zone of lesion the influence of chromatin structure and of the distance from damage are investigated. As first applications we demonstrate a locally confined, time‐dependent mobility increase of histone H1.2, and a progressive retardation of the DNA repair factor XRCC1 at damaged sites. This assay can be used to map the response of nuclear proteins to DNA damage in time and space. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

10H‐3,6‐Diazaphenothiazines triggered the mitochondrial‐dependent and cell death receptor‐dependent apoptosis pathways and further increased the chemosensitivity of MCF‐7 breast cancer cells via inhibition of AKT1 pathways

Breast cancer is one of the leading causes of death in cancer categories, followed by lung, colorectal, and ovarian among the female gender across the world. 10H‐3,6‐diazaphenothiazine (PTZ) is a thiazine derivative compound that exhibits many pharmacological activities. Herein, we proceed to investigate the pharmacological activities of PTZ toward breast cancer MCF‐7 cells as a representative in vitro breast cancer cell model. The PTZ exhibited a proliferation inhibition (IC₅₀ = 0.895 µM) toward MCF‐7 cells. Further, cell cycle analysis illustrated that the S‐phase checkpoint was activated to achieve proliferation inhibition. In vitro cytotoxicity test on three normal cell lines (HEK293 normal kidney cells, MCF‐10A normal breast cells, and H9C2 normal heart cells) demonstrated that PTZ was more potent toward cancer cells. Increase in the levels of reactive oxygen species results in polarization of mitochondrial membrane potential (ΔΨm), together with suppression of mitochondrial thioredoxin reductase enzymatic activity suggested that PTZ induced oxidative damages toward mitochondria and contributed to improved drug efficacy toward treatment. The RT² PCR Profiler Array (human apoptosis pathways) proved that PTZ induced cell death via mitochondria‐dependent and cell death receptor‐dependent pathways, through a series of modulation of caspases, and the respective morphology of apoptosis was observed. Mechanistic studies of apoptosis suggested that PTZ inhibited AKT1 pathways resulting in enhanced drug efficacy despite it preventing invasion of cancer cells. These results showed the effectiveness of PTZ in initiation of apoptosis, programmed cell death, toward highly chemoresistant MCF‐7 cells, thus suggesting its potential as a chemotherapeutic drug.     

Wavelength dependence of cell cloning efficiency after optical trapping.

A study on clonal growth in Chinese hamster ovary (CHO) cells was conducted after exposure to optical trapping wavelengths using Nd:YAG (1064 nm) and tunable titanium-sapphire (700-990 nm) laser microbeam optical traps. The nuclei of cells were exposed to optical trapping forces at various wavelengths, power densities, and durations of exposure. Clonal growth generally decreased as the power density and the duration of laser exposure increased. A wavelength dependence of clonal growth was observed, with maximum clonability at 950-990 nm and least clonability at 740-760 nm and 900 nm. Moreover, the most commonly used trapping wavelength, 1064 nm from the Nd:YAG laser, strongly reduced clonability, depending upon the power density and exposure time. The present study demonstrates that a variety of optical parameters must be considered when applying optical traps to the study of biological problems, especially when survival and viability are important factors. The ability of the optical trap to alter either the structure or biochemistry of the process being probed with the trapping beam must be seriously considered when interpreting experimental results.     

6‐O‐(3″, 4″‐di‐O‐trans‐cinnamoyl)‐α‐l‐rhamnopyranosylcatalpol and verbascoside: Cytotoxicity,cell cycle kinetics,apoptosis, and ROS production evaluation in tumor cells

The aim of this study was to evaluate the impact that 6‐O‐(3″, 4″‐di‐O‐trans‐cinnamoyl)‐α‐ l ‐rhamnopyranosylcatalpol (Dicinn) and verbascoside (Verb), two compounds simultaneously reported in Verbascum ovalifolium, have on tumor cell viability, apoptosis, cell cycle kinetics, and intracellular reactive oxygen species (ROS) level. At 100 µg/mL and 48 hours incubation time, Dicinn and Verb produced good cytotoxic effects in A549, HT‐29, and MCF‐7 cells. Dicinn induced cell‐cycle arrest at the G₀/G₁ phase and apoptosis, whereas Verb increased the population of subG₁ cells and cell apoptosis rates. Furthermore, the two compounds exhibited time‐dependent ROS generating effects in tumor cells (1‐24 hours). Importantly, no cytotoxic effects were induced in nontumor MCF‐10A cells by the two compounds up to 100 µg/mL. Overall, the effects exhibited by Verb in tumor cells were more potent, which can be correlated with its structural features, such as the presence of phenolic hydroxyl groups.