FTIR biospectroscopy investigation on cisplatin cytotoxicity in three pairs of sensitive and resistant cell line

Fourier Transformed Infrared Spectroscopy [FTIR] has extensively been used for biological applications. Cisplatin is one the most useful antineoplastic chemotherapy drugs for a variety of different human cancers. One of the clinical problems in its application, which would consequently affect the therapeutic outcome of its application, is the occurrence of resistance to this agent. In this project three different pairs of sensitive and resistant cell lines of human ovarian A2780 and its resistant pair of A2780-CP, human ovarian OV2008 and its resistant pair of C13, and finally human lung carcinoma of HTB56 and its resistant pair of HTB56-CP were grown in the laboratory under the standard procedure. Saline was exposed to control cells, whereas 1, 5 and 10 microg/ml of cisplatin was exposed to experimental cells, for one hour. Cells were then collected and lyophilized from which spectra were taken. According to our results, we could not trigger a well-recognized cells biomolecular band at 1015 cm[-1], being modified after exposure to cisplatin in all cell lines. On the other hand, there was a clear dose-dependent increase in protein beta-sheet structure related peaks shift in resistant cell lines after exposure to cisplatin. This would probably indicate an easier protein interaction site for cisplatin in the resistant cell lines, which would probably inhibit cisplatin from binding to DNA, as the cytotoxic target. As a conclusion, FTIR biospectroscopy has proven its potency to identify the interactions, as well as the false engagement cellular sites for cisplatin in sensitive and resistant cell lines

Immunomodulatory effects of bee venom in human synovial fibroblast cell line

As in Iranian traditional medicine, bee venom [BV] is a promising treatment for the rheumatoid arthritis [RA] which is considered as a problematic human chronic inflammatory disease in the present time. Smoking is considered to be a major risk factor in RA onset and severity. The main aim of this study is to investigate the effects of BV on cigarette smoke-induced inflammatory response in fibroblast-like synoviocytes [FLS]. Cytotoxicity of cigarette smoke condensate [CSC] and bee venom were determined by the tetrazolium [MTT] method in cultured synovial fibroblastes. The expression of interleukin-1 beta and sirtuin1 mRNA were analyzed by SYBR green real-time quantitative PCR. Differences between the mean values of treated and untreated groups were assessed by student t-test. Based on MTT assay, CSC and BV did not exert any significant cytotoxic effects up to 40 micro g/mL and 10 micro g/mL, respectively. Our results showed that interleukin-1 beta mRNA level was significantly up-regulated by CSC treatments in LPS-stimulated synoviocytes in a dose-dependent manner. Conversely, the expressions of IL-1 beta and Sirt1 were up-regulated even in lower concentrations of BV and attenuated at higher concentrations. Also, BV attenuated the CSC-induced and LPS-induced inflammatory responses in synovial fibroblasts. Our results support the epidemiological studies indicating pro-inflammatory effects of CSC and anti-inflammatory effects of BV on FLS cell line

Intracellular GSH alterations and its relationship to level of resistance following exposure to cisplatin in cancer cells

One of the major complications in cancer chemotherapy with cisplatin as one of the important medicines in treatment regimens of different cancers is the development of resistance. One of the most described cellular defense mechanisms involved in resistance is glutathione [GSH], thus in this study, the effects of cisplatin on the total intracellular GSH level [GSHi] in some sensitive and resistant variants of human cell lines [hepatocarcinoma HepG2, sking A375, cisplatin sensitive glioblastoma U373MG and cisplatin resistant glioblastoma U373MGCP, cisplatin sensitive ovary A2780S and cisplatin resistant A2780CP cells] were studied. MTT assay was performed to measure cytotoxicity of cisplatin [33.3 microM for 1 hour]. Following cisplatin exposure, GSHi [per million cells] was evaluated using a photometrical assay up to 90 minutes. Our results indicate that there are significant differences between GSHi content of A2780CP and U373MGCP cells compared to other cell lines. Moreover, IC[50] of cisplatin in different cells seems to have a relation with mean of GSH level in 90 minutes [GSH [mean][90]]. As a conclusion, it seems that resistance to cisplatin in different cell lines is more related with the diverse patterns of GSHi variations following cisplatin exposure than its original level, and/or its cellular increase or decrease. It is also suggested that GSH [mean][90] may be used as a factor for the prediction of cellular resistance to cisplatin

FTIR microspectroscopy reveals chemical changes in mice fetus following phenobarbital administration

Phenobarbital is a phenobarbiturate used as a sedative, anticonvulsant or hypnotic with the doses prescribed and can cause teratogenic effects. The goal of this study was to examine an alternative method for the recognition of the mechanism or the bimolecular potential changes in mice fetus caused by Phenobarbital using FTIR micro spectroscopy. The mice were injected with Phenobarbital [120 mg/Kg] on gestation day 9. Fetuses were dissected on day 15 of gestation and morphological and histological studies on the fetus were carried out. Sections [10 microm] of normal and Phenobarbital treated fetus brains and livers were used for FTIR measurement in the wave number region of 400- 4000 cm. The results were shown by 2 derivatization of spectra and also subtracting from control spectra. In liver, the intensity at 1054 cm, 1155 cm, 1353 cm, 1453cm,1645 cm, 1622 cm, 2944 cm, 2913 cm and 2845 cm were shifted and increased. In the brain, the intensity at 879 cm, 911 cm, 955 cm, 1223 cm, 1256 cm, 1304 cm, 1360 cm, 1453 cm, 1529 cm, 1636 cm, 2845 cm, 2915 cm and 2950 cm were increased and shifted. The most important changes of the fetus brain tissue are on the beta structure of proteins due to the amide I bands at 1636 cm, while extensive effects on the DNA structure were obvious for the Phenobarbital treated liver tissues. As a conclusion, FTIR spectroscopy might well be assumed as a potentially powerful teratogenic measurement instrument with a unique ability to identify the modified bimolecular structures

Toxicity effect of silver nanoparticles on mice liver primary cell culture and HepG2 cell line

Nano-silver [AgNP] has biological properties which are significant for consumer products, food technology, textiles and medical applications [e.g. wound care products, implantable medical devices, in diagnosis, drug delivery, and imaging]. For their antibacterial activity, silver nanoparticles are largely used in various commercially available products. Thus, the use of nano-silver is becoming more and more widespread in medicine. In this study we investigated the cytotoxic effects of AgNPs on liver primary cells of mice, as well as the human liver HepG[2] cell. Cell viability was examined with MTT assay after HepG[2] cells exposure to AgNPs at 1, 2, 3, 4, 5, 7.5, 10 ppm compared to mice primary liver cells at 1, 10, 50, 100, 150, 200, 400 ppm for 24h. AgNPs caused a concentration-dependent decrease of cell viability in both cells. IC50 value of 2.764 ppm [Micro g/ml] was calculated in HepG[2] cell line and IC[50] value of 121.7 ppm [Micro g/ml] was calculated in primary liver cells of mice. The results of this experiment indicated that silver nanoparticles had cytotoxic effects on HepG[2] cell line and primary liver cells of mice. The results illustrated that nano-silver had 44 times stronger inhibitory effect on the growth of cancerous cells [HepG[2] cell line] compared to the normal cells [primary liver cells of mice]. which might further justify AgNPs as a cytotoxic agents and a potential anticancer candidate which needs further studies in this regard

effects of extending of co-planarity in a series of structurally relative polypyridyl palladium[II] complexes on DNA-binding and cytotoxicity properties

In depth interaction studies between calf thymus deoxyribonucleic acid [CT-DNA] and a series of four structurally relative palladium[II] complexes [Pd[en][HB]][NO[3]][2] [a-d], where en is ethylenediamine and heterocyclic base [HB] is 2, 2›-bipyridine [bpy, a]; 1, 10-phenanthroline [phen, b]; dipyridoquinoxaline [dpq, c] and dipyridophenazine [dppz, d] [Figure 1], were performed. These studies have been investigated by utilizing the electronic absorption spectroscopy, fluorescence spectra and ethidium bromide [EBr] displacement and gel filtration techniques. a-d complexes cooperatively bind and denature the DNA at low concentrations. Their concentration at midpoint of transition, L1/2, follows the order a >> b > c > d. Also the g, the number of binding sites per 1000 nucleotides, follows the order a >> b c > d. EBr and Scatchard experiments for a-d complexes suggest efficient intercalative binding affinity to CT-DNA giving the order: d > c > b > a. Several binding and thermodynamic parameters are also described. The biological activity of these cationic and water soluble palladium complexes were tested against chronic myelogenous leukemia cell line, K562. b, c and d complexes show cytotoxic concentration [Cc[50]] values much lower than cisplatin

FTIR-microspectroscopy detection of metronidazole teratogenic effects on mice fetus

Metronidazole is used to treat trichomoniasis, bacterial vaginosis, and other diseases. There are controversy aspects about its teratogenicity. A teratogenic agent can alter morphology or subsequent function of the fetus. The aim of this study was to examine an alternative method for the recognition of the mechanism or the bimolecular potential changes in mice fetus caused by Metronidazole using FTIR micro spectroscopy. The mice were injected with metronidazole [60 mg/Kg] on gestation day 9. Fetuses were dissected on day 15 of gestation and morphological and histological studies on the fetus were carried out. Serial sectioning [10 micro m] of normal and metronidazole-treated brains and livers were used for FTIR measurement in the wave number region of 600- 3600 cm[-1].The results showed that there were some variations between the fetus of normal and treated brain and liver. The band intensities in fetus brain and liver of test animals were reduced and shifted at 707 cm[-1], 1155 cm[-1], 1054 cm[-1], 1256 cm[-1] and 1219 cm[-1], 1453 cm[-1] and 1525 cm[-1], 1622 cm[-1], 1645 cm[-1] and 2944 cm[-1],while the band intensities were increased and shifted at 879 cm[-1], 810 cm[-1], 1223 cm[-1], 1256 cm[-1] 1360 cm[-1], 1723 cm[-1]. It was concluded that most of variations in brain and liver of Metronidazole treated fetuses are in amid bands, nucleic acid and carbohydrate related bands. Based on these findings FTIR spectroscopy can be a useful tool for bio diagnostic

Anticancer Activity of Cobra Venom Polypeptide, Cytotoxin-II, against Human Breast Adenocarcinoma Cell Line (MCF-7) via the Induction of Apoptosis / 한국유방암학회지

PURPOSE: Breast cancer is a significant health problem worldwide, accounting for a quarter of all cancer diagnoses in women. Current strategies for breast cancer treatment are not fully effective, and there is substantial interest in the identification of novel anticancer agents especially from natural products including toxins. Cytotoxins are polypeptides found in the venom of cobras and have various physiological effects. In the present study, the anticancer potential of cytotoxin-II against the human breast adenocarcinoma cell line (MCF-7) was investigated. METHODS: The cytotoxic effects of cytotoxin-II were determined by morphological analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The mode and mechanism of cell death were investigated via acridine orange/ethidium bromide (AO/EtBr) double staining, flow cytometric analysis of cell death, detection of mitochondrial membrane potential, measurement of intracellular reactive oxygen species (ROS), annexin V/propidium iodide staining, and caspase-9 activity assays. RESULTS: The half maximal inhibitory concentration (IC50) of cytotoxin-II in MCF-7 cells was 4.18+/-1.23 microg/mL, while the value for cisplatin was approximately 28.02+/-1.87 microg/mL. Morphological analysis and AO/EtBr double staining showed typical manifestations of apoptotic cell death (in doses lower than 8 microg/mL). Dose- and time-dependent ROS generation, loss of mitochondrial membrane potential, caspase-9 activation, and cell cycle arrest were observed in their respective tests. CONCLUSION: In conclusion, cytotoxin-II has potent anticancer effects in the MCF-7 cell line, which are induced via the intrinsic pathways of apoptosis. Based on these findings, cytotoxin-II is a suitable choice for breast cancer treatment.

Cisplatin resistant patterns in ovarian cell line using FTIR and principle component analysis

Cisplatin is a common chemotherapeutic agent that used for treatment of many solid cancers. Rapid identification of chemotherapy resistance is very important and may lead to effective treatment plan. Spectroscopy techniques, such as infrared spectroscopy, which are sensitive to biochemical composition of samples, have shown potentials to discriminate tissues. Developing in Fourier transform infrared [FTIR] as a diagnostic tool support conventional technique in investigating cell phenotype. By this goal three different cell lines, two cisplatin resistant OV2008-DDP [C13] and A2780-CP ovarian cell lines and one cisplatin sensitive A2780 cell line were investigated by FTIR spectroscopy. Data were subjected to principle component analysis [PCA] to obtain FTIR pattern for cisplatin resistance. Using FTIR spectroscopy on these cells in the range of 400-4000 cm[-1] was shown dramatic change in cells. Results shows that Cisplatin resistance pattern is characterized in spectrum with the alteration of conformation in secondary structure of proteins and a shift toward the high wave numbers of CH2 stretching vibration. The FTIR data set between 1000 and 3000 cm[-1] could be consumed as biochemical typicality spectra among resistant and sensitive cell lines while correctly classified by PCA model. Our work supports the promise of PCA analysis of FTIR data as a powerful combined approach for the development of automated methods to recognize resistant to cisplatin in experimental cell lines. One of the advantages of this tool is to investigate the resistant percent of cancer cells .Such technique may bring new tool in cancer diagnosis and stage definition in cancerous tissues

Cytotoxic effects of two Iranian scorpions odontobuthusdoriae and bothutus saulcyi on five human cultured cell lines and fractions of toxic venom

Scorpion venom toxicity is of major concern due to its influence on human activities and public health. We investigated the in-vitro process of cell death caused by two Iranian scorpions Odontobuthus doriae and Bothutus salceyi venom on human cell lines. The aim of this study was to provide further information about triggering cell death and suggestion of methods for the elimination of unwanted cells such as tumor cells. The cytotoxicity and apoptosis induced by effect of scorpion venoms on five established eukaryotic cell lines are analyzed on different human cell lines. All cultured cell lines were incubated with varying doses of scorpion venom for 24 h at 37°C. Control culture was treated with an equal amount of SFM. The percentage of cell survival was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium [MTT] colorimetric assay. Our data demonstrated that Bothutus saulcyi, does not show cytotoxic effect on any of the used cell lines. Odontobuthus doriae, however, has resulted a dose dependent cytotoxic effect with maximum at 1 ug/mL on 1321N1 glioma like cell line. Then the cytotoxic venom of O. doriae was fractionated using Sephadex G50 gel chromatography. The toxic fractions on mouse used to Cytotoxicity assay on 1321 N1 cell line and data demonstrated that, the fraction F3 showed a dose dependent Cytotoxicity assay. Further studies to explode the mode of action of these venoms are recommended and purification of the toxic fraction should be done

Patterns prediction of chemotherapy sensitivity in cancer cell lines using FTIR spectrum, neural network and principal components analysis

Drug resistance enables cancer cells to break away from cytotoxic effect of anticancer drugs. Identification of resistant phenotype is very important because it can lead to effective treatment plan. There is an interest in developing classifying models of resistance phenotype based on the multivariate data. We have investigated a vibrational spectroscopic approach in order to characterize a sensitive human ovarian cell line, A2780, and its cisplatin-resistant derivative, A2780-cp. In this study FTIR method have been evaluated via the use of principal components analysis [PCA], ANN [artificial neuronal network] and LDA [linear discriminate analysis]. FTIR spectroscopy on these cells in the range of 400-4000 cm[-1] showed alteration in the secondary structure of proteins and a CH stretching vibration. We have found that the ANN models correctly classified more than 95% of the cell lines, while the LDA models with the same data sets could classify 85% of cases. In the process of different ranges of spectra, the best classification of data set in the range of 1000-2000 cm[-1] was done using ANN model, while the data set between 2500-3000 cm[-1] was more correctly classified with the LDA model. PCA of the spectral data also provide a good separation for representing the variety of cell line spectra. Our work supports the promise of ANN analysis of FTIR spectrum as a supervised powerful approach and PCA as unsupervised modeling for the development of automated methods to determine the resistant phenotype of cancer classification

Cytotoxicity of diimine palladium [II] complexes of alkyldithiocarbamate derivatives on human lung, ovary and liver cells

Three new Complexes of formula [pd[bpy][R-NH-CSS]] Cl [where bpy is 2/2'- bipyridine, and R-NH-CSS is butylamine, hexylamine- and octyamine-dithiocabamate anion] have been synthesized by University of Sistan and Blachostan. These complexes have been characterized by spectroscopic methods such as ultraviolet-visible, infrared and [1]H-NMR as well as conductivity measurements and chemical analysis. In these complexes, each of the dithiocarbamate ligands coordinates to Pd [II] center as bidentate with two sulfur atoms. We have found a 1:1 electrolyte in water conductivity test for the above mentioned compounds. To measure the biologic activity and potential anticancer efficacy of these compounds, they have been compared with cisplatin and its palladium analogue of [Pd [NH[3]][2] Cl[2]] on three different cell lines of human hepatocarcinoma HepG2, human ovarian carcinoma OV2008, and human lung adenocarcinoma A549. Clonogenic assay has shown LD[50]s in the range of 0.131 +/- 0.025 to 0.934 +/- 0.194 for these compounds on above cell lines. In comparison, cisplatin has shown LD[50]s of 0.838 +/- 0.074, 2.196 +/- 0.220, and 2.799 +/- 0.733 on OV2008, HepG2 and A549 cell lines, respectively. As a conclusion, above three new complexes have shown higher cytotoxicities compared to cisplatin on three different human cell lines. Based on biological tests, these compounds may potentially be considered as good anticancer candidates for further pharmacological studies

Evaluation of northern Iran mentha pulegium L cytotoxicity

In vitro tests could be a valuable tool for the evaluation of medicinal plants' cytotoxicity. One of the most frequently used Iranian traditional plants is Mentha Pulegium from Labiatae family. In the present study, essential oil and the methanolic extract of Mentha pulegium, were analyzed for cytotoxicity on human ovary adenocarcinoma SK-OV-3, human malignant cervix carcinoma Hela, and human lung carcinoma A549 cell lines. Two different assays of clonogenic and neutral red [NR] were used for evaluation of cytotoxicity. Although the methanolic extract of Mentha pulegium did not show any cytotoxic effects, the essential oil of this plant proved to be a potent cytotoxic agent on the above three cell lines. According to the clonogenic assay, LD50s of the essential oil on SK-OV-3, Hela and A549 cell lines are 14.10, 59.10 and 18.76 micro g/ml, respectively. Our findings suggest that Mentha pulegium essential oil might be considered as a potentially toxic agent on human cancer cell lines, and a possible candidate for human cancer chemotherapy. However, further biological tests on the efficacy and side effects of this plant are necessary before its use in human