Effect of dimethyl sulfoxide on mouse embryo fibroblasts: inhibition of plasminogen activator inhibitor deposition and interference with early events of serum-stimulated growth

Quiescent and serum-stimulated cultures of Swiss mouse embryo fibroblasts (MEF) showed alterations in cell morphology including an enlargement in size upon treatment with 2% dimethyl sulfoxide (DMSO). Treatment of MEF and monkey kidney epithelial cells (MK2) with 2% DMSO at the early periods of serum-stimulated growth inhibited RNA, protein and DNA synthesis. DMSO treatment of cells at late stages of serum-stimulated growth (MEF after 1 hr and MK2 cells after 3 hr of stimulation) had little effect on DNA and protein synthesis although cell enlargement occurred in these cells. When the [35S]methionine labelled proteins of the control and the DMSO treated cells were analysed by high resolution polyacrylamide gel electrophoresis, no apparent difference was observed in the pattern of intracellular proteins of these cells. In contrast, the extracellular levels of two serum-induced secreted proteins of MEF (Mr 48,000 and 26,000) were dramatically reduced by DMSO treatment. The DMSO sensitive 48 kDa protein was found to be the major component of the extracellular matrix, while the 26 kDa protein was not. The 48 kDa protein was identified as plasminogen activator inhibitor (PAI-1). Densitometric quantitation showed a gradual accumulation of this protein in the matrix of serum-stimulated cells. The deposition of this protein in the matrix was inhibited by DMSO. Flow-cytometric quantitation of indirect immunofluorescence indicated higher intracellular levels of the 48 kDa protein in fetal calf serum (FCS) + DMSO treated cells, suggesting that the low level of this protein in the medium of DMSO treated cells is probably due to lack of transport of this protein from the cells into the medium

Promoter hypermethylation profile of tumor-associated genes p16, p15, hMLH1, MGMT and E-cadherin in oral squamous cell carcinoma

Aberrant promoter hypermethylation of tumor-associated genes leading to their inactivation is a common event in many cancer types. Using a sensitive restriction-multiplex PCR method, we studied the promoter hypermethylation profile of the p16, p15, hMLH1, MGMT and E-cad genes in oral squamous cell carcinoma (OSCC) of Indians. We analyzed a total of 51 samples for the p15 tumor-suppressor gene and 99 samples for each of the remaining genes. Our studies indicate an incidence of promoter hypermethylation of 23% each for p16 and p15, 8% for hMLH1, 41% for MGMT and 35% for E-cad. We observed aberrant hypermethylation of the promoter region of at least 1 of these genes in 74.5% of cases (n = 51) for which all the 5 genes were studied. Abnormal methylation was detected in tumors irrespective of stage and location in the oral cavity, whereas no abnormal methylation was detectable in normal oral squamous tissues obtained from 25 OSCC patients. Detection of aberrant hypermethylation patterns of cancer-associated genes listed above is therefore suitable for diagnosis of OSCC in individuals at high risk for this disease

Cytotoxic and genotoxic effects of cleistanthin B in normal and tumour cells

Cleistanthin B, one of the toxic constituents of Cleistanthus collinus, was found to be cytotoxic to normal and tumour cells. In comparison with normal cells, tumour cells were sensitive to lower doses of toxin. The 50% growth inhibition (GI50) values for normal cell lines were from 2 × 10-5 to 4.7 × 10-4 M and for tumour cells the values ranged from 1.6 × l0-6 to 4 × l0-5 M. Short exposure (30 min) of Chinese hamster ovary (CHO) cells to cleistanthin B at 1-6 μg/ml resulted in extensive chromatid and isochromatid breaks and gaps. However there was no significant increase in cell death and DNA strand breaks in cells treated under the above conditions. Cleistanthin B induced micronucleus formation in cultured lymphocytes in a dose-dependent manner. CHO cells treated with high doses of cleistanthin B showed a decrease in cell viability and a concomitant increase in DNA strand-breaks. The cell death appears to be due to apoptosis since nucleosome-like ladders were observed in the treated cells when the DNA was electrophorized in agarose gels

Genomic instability and tumor-specific alterations in oral squamous cell carcinomas assessed by inter- (simple sequence repeat) PCR

Purpose: Genomic instability plays a major role in the genesis and progression of tumors, and in the evolution of tumor heterogeneity. To determine the role of genomic instability in the genesis and progression of oral cancer, we assessed the extent of genomic alterations in oral squamous cell carcinomas (OSCCs). Experimental Design: We used the recently developed inter-(simple sequence repeat) PCR technique to quantitate genomic instability using matched tumor and normal OSCC samples (n = 25). The inter-repeat region bands of similar molecular size observed to be altered in more than one case were sequenced and analyzed to identify probable OSSC-associated specific genetic lesions. Results: Of the four base-anchored, dinucleotide repeat-based primers used for the study, the most informative profile in OSCCs was generated by the (CA)8RG primer. Measurement of genomic instability index using the (CA)8RG primer revealed a high incidence of genomic instability in OSCCs. No significant correlation between the extent of alterations and stage or location of the tumor was observed. Sequencing analysis of the altered bands revealed gains/losses in several chromosomal regions. Of the matched tumor and corresponding normal tissue DNA studied, hitherto unreported losses were seen in 11p15 and 17q25 chromosomal regions. Sequencing of some of the tumor-specific altered regions indicated that they code for regions of UDP-GalNAc and hRAD 17 genes, which were lost (deleted) in oral cancer. Conclusions: Our results indicate that the extent of genomic instability in OSCC is not correlated to the tumor stage or location. For the first time, we have shown that chromosomal alterations detected by inter-(simple sequence repeat) PCR could be correlated to genes associated with cancer development

Cleistanthin A, a diphyllin glycoside from Cleistanthus collinus, is cytotoxic to PHA-stimulated (proliferating) human lymphocytes

An ideal anticancer drug would be one that preferentially kills tumor cells with the least toxicity to normal cells. Cleistanthin A, a diphyllin glycoside of the tropical plant Cleistanthus collinus, was found to possess cytotoxic and tumor regressing properties. To find out whether this compound acts selectively on proliferating cells it was tested against quiescent and proliferating human lymphocytes. Mitogen-stimulated and unstimulated human lymphocytes were treated with cleistanthin A. A cytotoxicity assay using MTT was used to assess the viability of the cells. Percentage viability of the unstimulated and treated cells were normalized to that of the untreated and unstimulated cells and percentage viability of stimulated and treated cells were normalized to that of stimulated and untreated cells. Quiescent lymphocytes were refractory to the action of cleistanthin A. Only proliferating cells were killed. Cell death was proportional to the percentage of cells in the proliferating stage and was also dose-dependent. Quiescent lymphocytes pretreated with cleistanthin A had the ability to proliferate upon subsequent stimulation with PHA. These results indicate that cleistanthin A does not affect the viability of quiescent cells. Also, it did not affect the proliferating potential of quiescent cells. However, this compound drastically affected proliferating cells by reducing their viability to 10-20%. Our results therefore indicate that the antiproliferative property of cleistanthin A could be used in regimens for treating tumors with extensive proliferative potencies

Inhibition of matrix metalloproteinase-9 (MMP-9) activity by cleistanthin A, a diphyllin glycoside from Cleistanthus collinus

Matrix metalloproteinases (MMPs) are responsible for the remodeling of the extracellular matrix throughout the body. Inhibitors of these enzymes have been suggested as potential therapeutic agents for use in treatment of cancer. Our observations showed that cleistanthin A, a diphyllin glycoside from the leaves of the plant Cleistanthus collinus inhibited MMP-9, a 92 kDa matrix metalloproteinase. Further evaluation of this compound might reveal many such novel properties that might help in the evolution of this compound as an anticancer drug

Genotoxicity of the herbicide butachlor in cultured human lymphocytes

Butachlor, a pre-emergence herbicide was investigated for its ability to induce sister chromatid exchanges (SCE) and chromosome aberrations (CA) in cultured human peripheral blood lymphocytes. Mitogen-stimulated lymphocytes were treated with three different concentrations (5, 10 and 20 μg/ml) of butachlor for 24, 48 and 72 h. Our results indicate a dose-dependent increase in the frequency of chromosomal aberrations at 24, 48 and 72 h of treatment with butachlor. No SCE was promoted by butachlor

SnCl₂-Catalyzed Selective Atom Economic Imino Diels–Alder Reaction: Synthesis of 2-(1<i>H</i>-Pyrrolo[2,3-<i>b</i>]pyridin-3-yl)quinolines

The synthesis of 2-(1<i>H</i>-pyrrolo­[2,3-<i>b</i>]­pyridin-3-yl)­quinolines by a SnCl₂-catalyzed multicomponent reaction has been described. The reaction proceeds chemo- and regioselectively in an atom-economic way, generating a library of 24 quinoline derivatives