KBr Cells Research Articles

Salubrinal increases the apoptosis of oral cancer cells by inhibiting radiation-induced activation of NF-Κb

Objective To explore the mechanism of the role of Salubrinal in regulating the radiation-induced apoptosis of oral cancer cells. Methods Radioresistant KBR cell line was constructed (4 Gy per fraction, every 7-10 d for 4 times). The radiosensitivity of oral cancer cells after Salubrinal pretreatment was measured by colony formation assay. The expression levels of NF-κB-HIF-1α signaling pathway and apoptosis biomarker cleaved PARP in oral cancer cells were measured by Western blot. The apoptosis rate was detected by Annexin V, PI staining and flow cytometry. Results Colony formation assay demonstrated that Salubrinal increased the radiosensitivity of oral cancer cells. The radiosensitization ratios of KB and KBR cells were 1.19 and 1.24. Western blot revealed that the activation of NF-κB-HIF-1α was time-dependent in the radiation-induced oral cancer cells, whereas Salubrinal inhibited the radiation-induced abnormal activation. In addition, Salubrinal increased the expression of apoptosis biomarker cleaved PARP and apoptosis index in radiation-induced oral cancer cells, whereas TNF-α, an activator of NF-κB, reversed the effect, suggesting that Salubrinal increased the apoptosis of radiation-induced oral cancer cells by suppressing the activation of NF-κB. Pretreatment of NF-κB inhibitor Bay11-7082 also increased the cell apoptosis. The expression levels of cleaved PARP of KB and KBR cell lines in the Bay11-7082+ IR group were 2.67±0.26 and 1.91±0.17, significantly higher compared with 2.1±0.16 and 1.44±0.15 in the IR group (both P<0.05). Conclusion Salubrinal can aggravate the apoptosis of radiation-induced oral cancer cells by inhibiting the radiation-induced activation of NF-κB, thereby regulating the radiosensitivity of oral cancer cells. Key words: Salubrinal; NF-κB; Oral cancer cell line; Apoptosis; Radiosensitivity

Inhibition of EGFR Abrogates Radioresistance in Human Oral Cancer by Inhibiting the Endoplasmic Reticulum Stress Chaperone GRP78

Combined radiotherapy and EGFR-targeted therapy regimen is preferred for patients with advanced oral cancer because it improves local cancer control and overall survival (OS) than radiotherapy alone. However, 50% of patients still develop local recurrence because of radioresistance. And some tumors showed radioresistance after inhibiting EGFR and the downstream signalling ERK, AKT,etc. Therefore, this study sought to identify new targets involved in resistance to EGFR-targeted therapy combined with radiotherapy.Endoplasmic reticulum stress (ERS) plays an important role in the regulation of radio- and chemoresistance in many malignancies. In this study, we investigated the role of EGFR and ERS signaling in the radioresistance of oral cancer. EGFR and ERS signalling pathway activation in KBoral carcinoma cells and the constructed corresponding radioresistant KBR cells after irradiation were detected by western blotting and real-time quantitative PCR. The radioresistance ofEGF-EGFR was detected using the colony formation assay. The cell apoptosis was detected by flow cytometry. And Cell proliferation was analysed using a Cell Counting Kit-8 (CCK-8) manufacturer’s protocol. DNA double-strand breaks and autophagy was detected by immunofluorescence. Immunohistochemistry was performed on 51 oral squamous cell carcinoma tissue specimens. The correlation between GRP78 and the prognosis of oral cancer patients was analysed using the Kaplan-Meier method. Herein, we constructed radioresistant oral cancer cells (KBR) and confirmed that EGF induced radioresistance, which could be reversed by silencing EGFR. Moreover, EGF activated radiation-induced ERS signalling PERK/ATF6/IRE1-GRP78 in oral cancer cells, whereas silencing EGFR inhibited the aforementioned functions. And EGF significantly increased the radiation-induced phosphorylation of ERK and AKT, while silencing PERK, IRE1α and ATF6 did not alter this effect. Consistent with others, ERS signalling in parallel with AKT/ERK in downstream of EGFR, and therefore may be targeted in amelioration of EGF-EGFR. In addition, silencing EGFR and the EGFR-targeted inhibitors cetuximab or nimotuzumab could inhibit radiation-induced DNA double-strand break (DSB) repair and autophagy, but increase radiation-induced cell apoptosis. The use of the ERS activators tunicamycin and thapsigargin could counteract this effect. Immunohistochemistry results showed that the ERS chaperone GRP78 was associated with poor prognosis of oral cancer (p < 0.05). These data indicate that inhibition of EGFR could reverse the radioresistance of oral cancer cells by inhibiting radiation-induced activation of ERSsignalling PERK/ATF6/IRE1-GRP78.

Comparative Analysis of Inkjet Printer Inks Extracted from Printed Documents by FT-IR Spectrophotometry

Primary coloured inks i.e. Cyan, Magenta, Yellow, and Black used in inkjet printers were extracted from the printed document and analyzed in order to identify the possible functional groups using Infrared (IR) spectroscopic technique. Experiments were conducted on the all four primary inks and the results are presented. The technique can provide valuable information if an admitted sample is provided for comparison with the suspect printed document. Determination of authenticity of a document is based on many factors. Characterization of writing/printing instruments used to produce the document such as ink; paper etc may be a leading step in the investigation. Many instrumental analytical techniques are available to determine the actual composition of dyes and other compounds present in inkjet printer inks. The IR technique is one of the techniques which can provides important information about the commercially available printer inks (Yuen et al, 2005). IR spectroscopy deals with the infra red region of the electromagnetic spectrum. The IR portion of the electromagnetic spectrum is usually divided into three regions; the near-IR (approximately 14000-4000 cm −1 ), mid-IR (4000-400 cm −1 ) and far- infrared (400-10 cm −1 ). The near-IR can excite overtone or harmonic vibrations, the mid-infrared may be used to study the fundamental vibrations and associated rotational- vibrational structure whereas the far-infrared has low energy and may be used for rotational spectroscopy. IR spectroscopy is based upon the ability of certain compounds to absorb radiation by interaction of IR radiation with the molecular bonds of the sample. Absorption occurs when the molecular bond has a vibrational frequency equal to the IR radiation. The wavelengths at which a measured absorption occurs correspond to specific functional groups. This information can be interpreted to characterize the compounds. Fourier Transform Infrared Spectroscopy (FTIR) is used to obtain an infrared spectrum of a solid, liquid or gas. In the Fourier Transform instrument the whole wavelength range is measured at once and then a transmittance or absorbance spectrum is generated with the help of a computer. Analysis of the position, shape and intensity of peaks in the spectrum reveals details about the molecular structure of the sample. A simple glass tube with length of 5 to 10 cm equipped with infrared windows at the both ends of the tube can be used for concentrations down to several hundred ppm. Sample gas concentrations well below ppm can be measured with a White's cell in which the infrared light is guided with mirrors to travel through the gas. Liquid samples can be sandwiched between two plates of highly polished transparent sodium chloride, potassium bromide or calcium fluoride. These plates are transparent to the infrared light. Solid samples can be prepared in a variety of ways such as by finely mixing the fine powder sample with an oily mulling agent (usually Nujol) in a marble or agate mortar, with a pestle. A thin of the mull is smeared onto salt plates and measured. The second method is to grind a quantity of the sample with potassium bromide finely. This powder mixture is then pressed in a mechanical press to form a translucent pellet through which the beam of the spectrometer can pass. A third technique is the cast film technique mainly used for polymeric materials and most suitable for qualititative analysis. The sample is first dissolved in a suitable, non hygroscopic solvent. A drop of this solution is deposited on surface of KBr or NaCl cell. The solution is then evaporated to dryness and the formed on the cell is analyzed directly. The final method is to use microtomy to cut a thin (20-100 µm) from a solid sample. IR technique can provide valuable information about the chemical composition of a material (Kan, et al., 2000; Wen, 2004; Huang, 2004) with convenient sample analysis.

Antitumor activity of satraplatin in cisplatin‐resistant oral squamous cell carcinoma cells

The aim of the current study was to identify the antitumor activity of satraplatin in paired cisplatin (CDDP)-resistant oral squamous cell carcinoma (OSCC) cell line and its parental cell line. CDDP-resistant (KB-R) cells and parental cells (KB) pair were used. Viability was assessed using the MTT and clonogenic assay. Real-time polymerase chain reaction (PCR), glutathione (GSH) assay, and flow cytometric analysis were used for further assessment. KB-R cells did not show cross-resistance to satraplatin. The expression status of almost all transporters was upregulated in the KB-R cells. There was no difference in the GSH levels between the KB and KB-R cells. Flow cytometric analysis indicated that with satraplatin the G2/M phase was arrested in the KB-R cells. KB-R cells contain enriched side population cells. These data suggested that satraplatin has antitumor activity against the CDDP-resistant OSCC cells. The mechanism of cross-resistance to platinum agents seems to be multifactorial.

Lipid rafts and metabolic energy differentially determine uptake of anti-cancer alkylphospholipids in lymphoma versus carcinoma cells

Perifosine is a member of the class of synthetic alkylphospholipids (APLs) and is being evaluated as anti-cancer agent in several clinical trials. These single-chain APLs accumulate in cellular membranes and disturb lipid-dependent signal transduction, ultimately causing apoptosis in a variety of tumor cells. The APL prototype edelfosine was previously found to be endocytosed by S49 mouse lymphoma cells via lipid rafts. An edelfosine-resistant cell variant, S49 AR, was found to be cross-resistant to other APLs, including perifosine. This resistance was due to defective synthesis of the raft constituent sphingomyelin, which abrogated APL cellular uptake. Sensitivity of S49 cells to edelfosine was higher than perifosine, which correlated with a relatively higher uptake. Human KB epidermal carcinoma cells were much more sensitive to APLs than S49 cells. Their much higher APL uptake was highly dependent on intracellular ATP and ambient temperature, and was blocked by chlorpromazine, independent of canonical endocytic pathways. We found no prominent role of lipid rafts for APL uptake in these KB cells; contrary to S49 AR cells, perifosine-resistant KBr cells display normal sphingomyelin synthesis, whereas APL uptake by the responsive KB cells was insensitive to treatment with methyl-β-cyclodextrin, a cholesterol-sequestrator and inhibitor of raft-mediated endocytosis. In conclusion, different mechanisms determine APL uptake and consequent apoptotic toxicity in lymphoma versus carcinoma cells. In the latter cells, APL uptake is mainly determined by a raft- and endocytosis-independent process, but metabolic energy-dependent process, possibly by a lipid transporter.

Optical constants and vibrational assignment of fluorobenzene between 4000 and 400 cm −1 at 25 °C

The optical constants (real and imaginary refractive indices), molar absorption coefficients, complex dielectric constants and complex molar polarizabilities of liquid fluorobenzene between 4000 and 400 cm −1 at 25 °C are reported for the first time. The complex refractive indices were determined from transmission measurements across the mid-infrared in KBr and NaCl cells with pathlengths from 2 to 3300 μm. The liquid phase fundamentals are assigned from the ν ˜ α ″ m spectrum with the aid of ab initio calculations and previous literature gas phase assignments of similar compounds.

Infrared Absorption Characteristics of Large-Sized Spherical Aryl-Ether Dendrimers

A specially designed experimental setup including a newly constructed KBr cell, a light source, a monochromator and an IR detection system suitable for strictly calibrated IR absorption measurements was constructed in order to elucidate strange phenomenon associated with cis-trans photo-isomerization at 6.26 µm of the azobenzene core located at the center of an aryl-ether dendrimer (L5AZO). The setup used an ordinary Nichrome source to measure the photon flux, irradiation area, and photon absorption rate as exactly as possible. The photon absorption rate was obtained as 1.2 × 10–3 photons/s·molecule and the absorption cross section of L5AZO molecules was estimated to be 3.1 × 10–19 cm2/molecule. Since these values of the absorption rate and absorption cross section are not abnormal, the occurrence of a simultaneous five-photon absorption is almost impossible. Therefore, the five-photon absorption and photo-isomerization shown by L5AZO suggested that the multiphoton process was not simultaneous absorption, but possibly a sequential absorption and energy storage until the equivalent energy of isomerization was reached. Japan Science and Technology Corporation, p]Japan

Bcl-2 plays a key role instead of mdr1 in the resistance to hexadecylphosphocholine in human epidermoid tumor cell line KB

We induced tolerance to hexadecylphosphocholine (HePC) in the human epidermoid tumor cell line, KB. After 70 weeks of adaptation, the IC 50 of HePC in the resistant cells KBr was 32-fold higher than in parental KB cells, and they were 30-fold more resistant to another ether lipid analogue, ET-18-OCH 3. The KBr cells also showed cross-resistance to vincristine and colchicine while remaining sensitive to other chemotherapy agents. RT–PCR assays showed that expression of the multidrug resistance gene (MDR1) was positive in KBr cells, whereas the expression of GST-pi (glutathione S-transferase pi) and MRP (multidrug resistance protein) was undetectable in KBr cells. Both an immunocytochemistry test and Western blot analysis indicated that the expression of bcl-2 in KBr cells was strongly positive, while it was only mildly expressed in KB cells. Verapamil could not reverse the resistance of KBr to HePC although it is a well-known reversing agent against MDR1. Our results suggest that bcl-2 instead of MDR1 plays a major role in the resistance of KBr cells.

Induction of resistance to hexadecylphosphocholine in the highly sensitive human epidermoid tumour cell line KB

Hexadecylphosphocholine (HePC, Miltefosine) is a representative of the group of alkyl-lysophosphocholines showing remarkable antitumoral activity in in vitro experiments and in experimental animal tumour models. The epidermoid tumour cell line KB, which is highly sensitive to HePC (half-maximal growth inhibiting concentration, ic 50: 1.2 μM; half lethal concentration, lc 50: 2.8 μM), was slowly adapted to increasing concentrations of HePC. After 14 months, the adaptation process was stopped at a concentration of 10 μg/ml (23.5 μM). At this point, the KB cells tolerated high doses of HePC ( ic 50: 41.2 μM; lc 50: 87.1 μM). The resistant cells (KBr) also showed crossresistance to the other well studied ether-lysophospholipids, Edexfosine ( 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, OMG-3PC; ET18OCH3) and Ilmofosine (1 S-hexadecyl-2-methoxymethyl-rac-(1-thio-3-hydroxy)propyl-3-phosphocholine, BM 41.440). Comparison of the KB and KBr cells showed that total lipid phosphate, ether-lipid content, vinyl-ether-lipid content, protein content as well as cholesterol content were unchanged. Furthermore, no changes were observed in the lipid composition between KB and KBr cells. Uptake of choline was also unchanged in both cells, but the uptake of d-myo-inositol was lower by a factor of two in the KBr cells. However, in KB cells, the addition of HePC induced a 50% reduction of d-myo-inositol-uptake, whereas in KBr cells inositol uptake was unchanged. Differences in HePC uptake and HePC metabolism were apparent between the KB and KBr cell lines. KBr cells showed a 3-fold lower uptake for HePC and a 3- to 4-fold faster metabolism of HePC than KB cells. However, the amount of non-metabolised HePC after 2 days of incubation with 1 μg/ml HePC ( lc 50: 1.2 μg/ml) in KB cells was 3- to 4-fold lower than the amount of HePC in KBR cells at 10 μg/ml ( lc50: 37 μg/ml), indicating that KBr cells can incorporate higher amounts of HePC than KB cells without adverse effects for cell growth and viability. This seems to indicate that mechanisms other than slower uptake and faster metabolism are involved in the induction of resistance to HePC in KBr cells.

Effects of hexadecylphosphocholine on membrane phospholipid metabolism in human tumour cells.

Hexadecylphosphocholine (HePC) is an analogue of the antiproliferative alkyllysophospholipids (ALP). As these lipid-like compounds interfere with membrane lipid metabolism at several sites, we studied the effects of HePC on uptake and metabolism of inositol and choline, two important phospholipid precursor molecules in two sensitive cell lines, Raji and KB, and in a resistant variant of KB cells, KBr. HePC substantially inhibited the membrane uptake of inositol and of choline in KB and Raji. Inositol uptake of KBr cells was constitutively low and was not further decreased by HePC. In all three cell lines, uptake inhibition of choline was less pronounced. Uptake inhibition showed characteristics of a non-specific effect, probably due to the physicochemical properties of HePC as a "lyso" structure. Decreased uptake of inositol did not affect phosphoinositide synthesis. Cellular phosphatidylcholine (PC) metabolism seemed to be affected through inhibition of choline incorporation and enhancement of PC degradation in the two sensitive cells. In KBr cells, these effects were not observed.

Very mild interconversion between aminoacetonitrile and the interstellar species methanimine and hydrogen cyanide

Methanimine 2 and hydrogen cyanide 3 were detected at RT in the microwave spectrum of aminoacetonitrile 1 . As 2 and 3 react together in the condensed phase, on a KBr cell cooled at very low temperature, an important role of 1 in interstellar chemistry may be postulated.

Electronic, counterion and medium effects on the bonding mode of the seleno-cyanate ion in trans-hydridoselenocyanatotetrakispiperidineiriduium(III) complexes

The results of infrared spectral studies of the complexes trans-[Ir(pip) 4(H)CNSe]Y (pip = piperidine; CNSe − = selenocyanate ion, bonding mode unspecified; Y = NCSe −, B(C 6H 5) − 4, and ClO − 4), both in the solid state and in solution, having shown them to be ideal systems for the investigation of ambidentate ligand bonding mode controls. The greater stability of the N-bound selenocyanate, with SeCN − as the counterion, both in the solid state and in solution (irrespective of solvent dielectric), serves to further confirm the existence of antisymbiosis in an octahedral complex of a class (b) metal involving a strong trans-director (H −) with no π-bonding capability. The greater stability of the Se-bound selenocyanate, with B(C 6H 5) − 4 as the counterion, in the solid state is a clear-cut example of counterion bonding mode control. The selenocyanato—tetraphenylborate complex was found to be subject to a KBr-assisted isomerization in the solid state, and all three complexes were observed to undergo a heterogeneous KBr-catalyzed ionization of the coordinated SeCN − ion in DMSO solution, when studied in KBr cells. In CH 2Cl 2 solution, where minimal KBr catalysis is observed, hydride transfer to the solvent, with a concomitant loss of NCSe − to give a five-coordinate intermediate, which recombines with NCSe − to form trans-[Ir(pip) 4(SeCN) 2] +, occurs instead over an extended time period. The vCN frequency frequency of the non-coordinated NCSe − is very sensitive to the solvent dielectric, suggesting the existence of H-bonding to the amine proton of the piperidine ligands in the low dielectric solvents. The complexes trans-[Ir(pip) 4(H)X] ClO 4 (X = NCS −, CN −) were included in the study, for purposes of comparison. The vIrH frequency exhibited by the cyanide complex was found to be very low, due to the strong trans-influence of the cyanide group.

Response of cultured mammalian cells to diphtheria toxin. V. Concurrent resistance to ribonucleic acid viruses in diphtheria toxin-resistant KB cell strains.

It was determined that KB-R cell strains, isolated from the KB cell line and resistant to diphtheria toxin, also resist infection by poliovirus, Mengo virus, vesicular stomatitis virus, and Newcastle disease virus. This resistance manifests itself by reduction in yields of progeny virus (a reduction of more than 2 logs in some cases), reduced production of viral-specific ribonucleic acid (RNA), and delayed cytopathic effect. In three KB-R strains tested, resistance was related to a step which falls between adsorption of virus and uncoating or release of viral messenger RNA. In two of these three strains, a second resistance mechanism was also active, causing a reduced production of viral-specific RNA. A relationship between the resistance to diphtheria toxin and the resistance to viral infection of the KB-R strains is considered. It has been postulated that the native diphtheria toxin molecule must be "activated" at the surface of a susceptible cell by a proteolytic process before it can enter the cell and inhibit protein synthesis. It is also known that the eclipse of some viruses occurs at or near the cell membrane and involves proteolytic activity. Resistance to viruses and toxin in the KB-R strains may result from the loss or modification of related labilizing or activating principles associated with the surface receptors for these agents.