Salivary Raman Spectroscopy: Standardization of Sampling Protocols and Stratification of Healthy and Oral Cancer Subjects.

Minimally invasive cancer detection using bio-fluids has been actively pursued due to practical limitations, though there are better suited noninvasive and online in vivo methods. Saliva is one such clinically informative bio-fluid that offers the advantages of easy and multiple sample collection. Despite its potential in cancer diagnostics, saliva analysis is challenging due to its heterogeneous composition. Recently, there has been an upsurge in saliva exploration using optical techniques. Forms of saliva such as precipitate and supernatant have been monitored, but this sampling method needs to be standardized due to the obvious loss of analytes in processing. In that context, present work details the comparison of four different saliva sampling methodologies, i.e., air-dried, lyophilized, pellet, and supernatant using Raman spectroscopy collected from 10 healthy samples. Composition-driven spectral features of all forms were compared and classified using principal component analysis and linear discriminant analysis. Analysis was carried out on all four groups in the first step. In the second step, groups of pellet and supernatant , and air-dried and lyophilized were analyzed. Findings suggest that pellet and supernatant exhibit discrete spectroscopic features and demonstrate high classification efficiency, which is indicative of their distinctive biochemical composition. On the other hand, air-dried and lyophilized forms showed overlapping spectral features and low classification, suggesting these forms retain majority spectroscopic features of whole saliva and are less prone to sampling losses. Thus, this study indicates air-dried and lyophilized forms may be more appropriate for saliva sampling using Raman spectroscopy providing the comprehensive information required for cancer diagnosis. Furthermore, the method was also tested for the classification of oral cancer and healthy subjects (n = 27) which yielded 90% stratification. The findings of the study indicate the utility of minimally invasive salivary Raman-based diagnostics in oral cancers.

Allicin Overcomes Hypoxia Mediated Cisplatin Resistance in Lung Cancer Cells through ROS Mediated Cell Death Pathway and by Suppressing Hypoxia Inducible Factors.

BACKGROUND/AIMS: The hypoxic microenvironment in NSCLC has been widely accepted as a contributor to both therapeutic resistance and tumor progression. In this study, we have explored Allicin, a key organosulfur compound present in garlic for its previously unreported effectiveness in the heterogeneous hypoxic tumor microenvironment of NSCLC. METHODS: The effect of Allicin on the viability of NSCLC cells was determined by MTT assay. To determine the migration rate of treated cells compared to the control, scratch and transwell migration assays were performed. Flowcytometry was done to explore cell cycle distribution, apoptosis and ROS production in cells. Fluorescence microscopy was used to examine autophagy and DNA damage in cells. Dot blot was done to check genome wide methylation. RNA expression was detected by RT-PCR and protein expression by western blotting. RESULTS: Allicin significantly decreases cell viability, proliferation and migration of NSCLC cells in both normoxia and hypoxia. It elicits both apoptosis and autophagy pathway in A549 cells by ROS accumulation and facilitating S/G2-M phase arrest in both normoxia as well as hypoxia. We suggest that ROS/MAPK and ROS/JNK signaling pathway together govern the cytotoxic effect of allicin in NSCLC cells. Notably, allicin suppresses the expression of HIF-1α and HIF-2α in hypoxic cells, pointing towards a mechanism of its effectiveness in hypoxia. A long term passive demethylation was observed, with decreased mC and no change in TET expression, thereby ruling out active demethylation by allicin. Furthermore, allicin synergistically enhances growth inhibitory activity of low dose cisplatin to effectively overcome hypoxia induced cisplatin resistance in A549 cells. CONCLUSION: Altogether, our results elucidate a potential use of allicin in sensitizing hypoxic and chemoresistant NSCLC to cisplatin-based chemotherapy and provide new, affordable therapeutic strategy with reduced side effects.

Raman exfoliative cytology for oral precancer diagnosis.

Oral premalignant lesions (OPLs) such as leukoplakia, erythroplakia, and oral submucous fibrosis, often precede oral cancer. Screening and management of these premalignant conditions can improve prognosis. Raman spectroscopy has previously demonstrated potential in the diagnosis of oral premalignant conditions (in vivo), detected viral infection, and identified cancer in both oral and cervical exfoliated cells (ex vivo). The potential of Raman exfoliative cytology (REC) in identifying premalignant conditions was investigated. Oral exfoliated samples were collected from healthy volunteers (n=20), healthy volunteers with tobacco habits (n=20), and oral premalignant conditions (n=27, OPL) using Cytobrush. Spectra were acquired using Raman microprobe. Spectral acquisition parameters were: λex: 785 nm, laser power: 40 mW, acquisition time: 15 s, and average: 3. Postspectral acquisition, cell pellet was subjected to Pap staining. Multivariate analysis was carried out using principal component analysis and principal component-linear discriminant analysis using both spectra- and patient-wise approaches in three- and two-group models. OPLs could be identified with ∼77% (spectra-wise) and ∼70% (patient-wise) sensitivity in the three-group model while with 86% (spectra-wise) and 83% (patient-wise) in the two-group model. Use of histopathologically confirmed premalignant cases and better sampling devices may help in development of improved standard models and also enhance the sensitivity of the method. Future longitudinal studies can help validate potential of REC in screening and monitoring high-risk populations and prognosis prediction of premalignant lesions.

Structural-conformational aspects of tRNA complexation with chloroethyl nitrosourea derivatives: A molecular modeling and spectroscopic investigation.

Chloroethyl nitrosourea derivatives (CENUs) represent an important family of anticancer chemotherapeutic agents, which are used in the treatment of different types of cancer such as brain tumors, resistant or relapsed Hodgkin's disease, small cell lung cancer and malignant melanoma. This work focuses towards understanding the interaction of chloroethyl nitrosourea derivatives; lomustine, nimustine and semustine with tRNA using spectroscopic approach in order to elucidate their auxiliary anticancer action mechanism inside the cell. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), Fourier transform infrared difference spectroscopy, circular dichroism spectroscopy and UV-visible spectroscopy were employed to investigate the binding parameters of tRNA-CENUs complexation. Results of present study demonstrate that all CENUs, studied here, interact with tRNA through guanine nitrogenous base residues and possibly further crosslink cytosine residues in paired region of tRNA. Moreover, spectral data collected for nimustine-tRNA and semustine-tRNA complex formation indicates towards the groove-directed-alkylation as their anti-malignant action, which involves the participation of uracil moiety located in major groove of tRNA. Besides this, tRNA-CENUs adduct formation did not alter the native conformation of biopolymer and tRNA remains in A-form after its interaction with all three nitrosourea derivatives studied. The binding constants (Ka) estimated for tRNA complexation with lomustine, nimustine and semustine are 2.55×102M-1, 4.923×102M-1 and 4.223×102M-1 respectively, which specify weak type of CENU's binding with tRNA. Moreover, molecular modeling simulations were also performed to predict preferential binding orientation of CENUs with tRNA that corroborates well with spectral outcomes. The findings, presented here, recognize tRNA binding properties of CENUs that can further help in rational designing of more specific and efficient RNA targeted chemotherapeutic agents.

tRNA binding with anti-cancer alkaloids-nature of interaction and comparison with DNA-alkaloids adducts.

Vincristine and vinblastine are potent anti-proliferative compound whose mechanism of action inside a cell is not well elucidated and the basis of their differential cellular effect is also unknown. This work focuses towards understanding the interaction of vincristine and vinblastine with tRNA using spectroscopic approach. Fourier transform infrared (FTIR) spectroscopy, Fourier transform infrared difference spectroscopy and UV-visible spectroscopy were used to study the binding parameters of tRNA-alkaloids interaction. Both the vinca alkaloids interact with tRNA through external binding with some degree of intercalation into the nitrogenous bases. The alkaloids adduct formation did not alter the A-conformation of the biopolymer and vincristine-tRNA complexes were found to be more stable than that of vinblastine-tRNA complexes. The binding constants (K) estimated for VCR-tRNA and VBS-tRNA complexation are 3×10(2)M(-1) and 2.5×10(2)M(-1) respectively, which suggests low affinity of alkaloids to tRNA. The study recognizes tRNA binding properties of vital vinca alkaloids and contributes to a better understanding of their mechanism of action and could also help in identifying the reason behind their diverse action in a cell.

Nucleic acid binding properties of allicin: spectroscopic analysis and estimation of anti-tumor potential.

BACKGROUND: Allicin has received much attention due to its anti-proliferative activity and not-well elucidated underlying mechanism of action. This work focuses towards determining the cellular toxicity of allicin and understanding its interaction with nucleic acid at molecular level. METHODS: MTT assay was used to assess the cell viability of A549 lung cancer cells against allicin. Fourier transform infrared (FTIR) and UV-visible spectroscopy were used to study the binding parameters of nucleic acid-allicin interaction. RESULTS: Allicin inhibits the proliferation of cancer cells in a concentration dependent manner. FTIR spectroscopy exhibited that allicin binds preferentially to minor groove of DNA via thymine base. Analysis of tRNA allicin complex has also revealed that allicin binds primarily through nitrogenous bases. Some amount of external binding with phosphate backbone was also observed for both DNA and RNA. UV visible spectra of both DNA allicin and RNA allicin complexes showed hypochromic shift with an estimated binding constant of 1.2×10(4)M(-1) for DNA and 1.06×10(3)M(-1)for RNA binding. No major transition from the B-form of DNA and A-form of RNA is observed after their interaction with allicin. CONCLUSIONS: The results demonstrated that allicin treatment inhibited the proliferation of A549 cells in a dose-dependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin. GENERAL SIGNIFICANCE: The results describe cytotoxic potential of allicin and its binding properties with cellular nucleic acid, which could be helpful in deciphering the complete mechanism of cell death exerted by allicin.

Spectroscopic and molecular docking studies on chlorambucil interaction with DNA.

Chlorambucil (CMB) is an anticancer drug used for the treatment of variety of cancers. Structural and conformational changes associated with DNA after binding with CMB were explored using spectroscopic techniques to get insight into the mechanism of action of CMB at molecular level. Different molar ratios of CMB-DNA complex were prepared with constant DNA concentration under physiological conditions. FTIR spectroscopy, UV-visible spectroscopy, CD spectroscopy and molecular docking studies were employed to determine the binding site and binding constant of CMB with DNA. The results show CMB binds DNA through nitrogenous bases (thymine, guanine and cytosine). The binding constant was calculated to be 1.3 × 10³ M⁻¹, which suggests weak binding of CMB with DNA double helix. FTIR and CD results show that CMB do not disturb native B-conformation of DNA and it continues to remain in its B conformation even at higher concentrations of CMB. The molecular docking results are in corroboration with our experimental results and provides structural insight into the interaction site.

Binding of an indole alkaloid, vinblastine to double stranded DNA: a spectroscopic insight in to nature and strength of interaction.

Vinblastine is a chemotherapeutic drug, used for the treatment of various cancers. It functions by interfering with DNA in fast growing cells and preventing them from reproducing. The present work is focused on the interaction of vinblastine with double stranded DNA in aqueous solution. Fourier transform infrared and UV-Visible absorption spectroscopy were used to analyze the interaction of vinblastine with calf-thymus DNA. FTIR analysis showed binding of vinblastine through A-T and G-C base pairs of DNA along with its phosphate backbone. UV-Vis spectroscopy results suggested the intercalation of drug in between the base pairs of DNA double helix. The binding constant estimated for vinblastine-DNA association was found to be K=1.7×10(3)M(-1). Molecular docking was performed and the results showed adenine base binding of vinblastine with DNA. Furthermore spectroscopic results revealed that formation of vinblastine-DNA complex resulted in no major change in the B-conformation of DNA.

Analysis of ovarian tumor pathology by Fourier Transform Infrared Spectroscopy.

BACKGROUND: Ovarian cancer is the second most common cancer among women and the leading cause of death among gynecologic malignancies. In recent years, infrared (IR) spectroscopy has gained attention as a simple and inexpensive method for the biomedical study of several diseases. In the present study infrared spectra of normal and malignant ovarian tissues were recorded in the 650 cm-1 to 4000 cm-1 region. METHODS: Post surgical tissue samples were taken from the normal and tumor sections of the tissue. Fourier Transform Infrared (FTIR) data on twelve cases of ovarian cancer with different grades of malignancy from patients of different age groups were analyzed. RESULTS: Significant spectral differences between the normal and the ovarian cancerous tissues were observed. In particular changes in frequency and intensity in the spectral region of protein, nucleic acid and lipid vibrational modes were observed. It was evident that the sample-to-sample or patient-to-patient variations were small and the spectral differences between normal and diseased tissues were reproducible. CONCLUSION: The measured spectroscopic features, which are the spectroscopic fingerprints of the tissues, provided the important differentiating information about the malignant and normal tissues. The findings of this study demonstrate the possible use of infrared spectroscopy in differentiating normal and malignant ovarian tissues.

DNA interaction studies of an anticancer plant alkaloid, vincristine, using Fourier transform infrared spectroscopy.

The binding of vincristine with DNA has been investigated using Fourier transform infrared spectroscopy. Various changes in the double helical structure of DNA after addition of vincristine have been examined. It is evident from Fourier transform infrared results that vincristine-DNA interaction occurs through guanine and cytosine base pairs. External binding of vincristine with phosphate backbone of the DNA is also observed. Vincristine perturbs guanine band at 1714 cm(-1), cytosine band at 1488 cm(-1), and the phosphate vibrations at 1225 and 1086 cm(-1). The UV-visible spectra of vincristine-DNA complex show hypochromic and bathochromic shifts, indicating the intercalation of vincristine into the double helical structure of DNA. Both intercalative and external binding modes are observed for vincristine binding with DNA, with an estimated binding constant K = 1.0 × 10(3) M(-1).

Thermal stability studies of 5-fluorouracil using diffuse reflectance infrared spectroscopy.

5-Fluorouracil is one of the oldest chemotherapy drugs and it has been in use for decades. It is an active medicine against several types of cancer and effectively blocks the replication of DNA viruses. The present study assessed the potential of diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy to determine the thermal stability of 5-fluorouracil. Infrared spectra of the drug before and after exposure to thermal radiation at different temperatures were collected in diffuse reflectance mode using a Fourier transform infrared (FTIR) spectrophotometer. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analysis were carried out simultaneously to confirm and support the results of infrared spectroscopy. The DRIFT spectra reveal that the drug shows good thermal stability up to 275 degrees C and undergoes complete thermal breakdown at about 285 degrees C. The results of DSC and XRD analysis also give the same information, which support the implementation of diffuse reflectance infrared spectroscopy for the determination of thermal stability of 5-fluorouracil.