Sesquiterpene lactones isolated from Elephantopus scaber L. inhibits human lymphocyte proliferation and the growth of tumour cell lines and induces apoptosis in vitro.

This study was designed to isolate the compounds responsible for the cytotoxic properties of South Indian Elephantopus scaber L. and further investigate their effects on quiescent and proliferating cells. Bioassay-guided isolation of the whole plant of chloroform extract of South Indian Elephantopus scaber afforded the known sesquiterpene lactone, deoxyelephantopin, and isodeoxyelephantopin whose structures were determined by spectroscopic methods. These compounds caused a dose dependent reduction in the viability of L-929 tumour cells in 72 h culture (IC(50) value of 2.7 µg/mL and 3.3 µg/mL) by the cell viability assay. Both the compounds act selectively on quiescent and PHA-stimulated proliferating human lymphocytes and inhibited tritiated thymidine incorporation into cellular DNA of DLA tumour cells. The compound deoxyelephantopin at a concentration of 3 µg/mL caused maximum apoptotic cells. It also exhibited significant in vivo antitumour efficacy against DLA tumour cells. The results, therefore, indicate that the antiproliferative property of deoxyelephantopin and isodeoxyelephantopin could be used in regimens for treating tumors with extensive proliferative potencies.

Evaluation of Elephantopus scaber on the inhibition of chemical carcinogenesis and tumor development in mice.

The effect of the active fraction of Elephantopus scaber L. (Asteraceae) (ES) on skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) as an initiator and croton oil as promoter was studied in mice. The active fraction of E. scaber (100 mg/kg) on topical application delayed the onset of papilloma formation and reduced the mean number of papillomas and the mean weight of papillomas per mouse. The intraperitoneal administration of the active fraction of E. scaber also had a significant effect on subcutaneous injection of 20-methylcholanthrene (20-MCA)-induced soft tissue sarcomas in mice. It inhibited the incidence of sarcomas and reduced the tumor diameter compared to MCA-treated control animals. The subcutaneous administration of the active fraction of E. scaber significantly inhibited the growth of subcutaneously transplanted DLA and EAC solid tumors, delayed the onset of tumor formation, and increased the life span of tumor bearing mice. The present study thus indicates the tumor inhibitory activity of the active fraction of E. scaber against chemically induced tumors and its ability to inhibit the development of solid tumors.

Cytotoxicity Profiling of Annona Squamosa in Cancer Cell Lines.

Objective: In the study our aim was to evaluate the cytotoxic activity of different solvent extracts of Annona squamosa seeds. Methods and materials: The four extracts used were petroleum ether, chloroform, ethyl acetate and methanol were tested using cytotoxicity assays. Results: Among the four extracts tested petroleum ether showed maximum cytotoxicity against a panel of cancer cell lines such as nasopharyngeal cancer (KB) cells, lung cancer (A-549) cells, breast cancer (MCF- 7) cells, leukemic (K-562) cells and inhibited the growth of murine cancer cells such as Dalton's lymphoma ascites (DLA) and Ehrlich ascites carcinoma (EAC). Conclusion: Petroleum ether extract of Annona squamosa seeds showed cytotoxicity towards a panel of cancer cells meanwhile non-significant cytotoxicity towards normal cells.

Anti-metastatic effect of deoxyelephantopin from Elephantopus scaber in A549 lung cancer cells in vitro.

In this study, we focused on the in vitro anti-metastatic effects of deoxyelephantopin (DOE), a sesquiterpene lactone from Elephantopus scaber on lung cancer A549 cells. DOE significantly decreased the metastatic potential of A549 cells as demonstrated by transwell invasion and migration assay. DOE inhibited the expression of matrix metalloproteinase-2 (MMP-2), MMP-9, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor at transcript level. Tissue inhibitors of metalloproteinase-2 (TIMP-2) mRNA levels was up-regulated in A549 tumour cells without any change in TIMP-1 expression after DOE treatment. DOE inhibited the protein levels of p-ERK1/2 and p-Akt in A549 cells but it activated p-JNK, p-p38 protein expression. NF-κB and IκBα expressions were down-regulated in DOE-treated cells. All these results demonstrated that DOE has shown anti-metastatic activity against A549 tumour cells.

Influence of plasma GSH level on acute radiation mucositis of the oral cavity.

PURPOSE: To see how pretreatment plasma GSH level influences the severity of acute radiation mucositis of the oral cavity during therapeutic irradiation in patients with oral cancer. METHODS AND MATERIALS: Thirteen patients with squamous cell carcinoma of the oral cavity form the subject material. Radical radiotherapy (60 Gy in 25 fractions over 5 weeks) was given using telecobalt. Pretreatment plasma GSH level was measured by Beutler's method. The normal tissue reaction during radiotherapy was monitored and graded. RESULTS: The GSH levels ranged from 10.6-90.5 microM/L (mean 30.6 microM/L). Those who had higher GSH levels developed less severe mucositis. The mean GSH levels in the groups with different severity of reactions were: Grade 2 (four patients) = 50.7 microM/L; Grade 3 (five patients) = 26.1 microM/L; Grade 4 (two patients) = 20.4 microM/L and Grade 5 (two patients) = 26.1 microM/L; Grade 4 (two patients) = 20.4 microM/L and Grade 5 (two patients) = 13.6 microM/L. CONCLUSION: Plasma GSH estimation has the potential to predict individual sensitivity to acute radiation mucositis and may particularly be useful in hyperfractionated regimes. The study also affirms the radioprotective role of GSH and suggests that this effect is either due to protection against membrane lipid peroxidation (since GSH does not enter the cell freely) or DNA damage (fractionated radiotherapy may permit freer entry of GSH into cell).

Circulating immune complexes as an immunological marker in premalignant and malignant lesions of the oral cavity.

Circulating immune complexes and their immunoglobulin contents were estimated in the sera of 50 patients with oral leukoplakia, 50 patients with oral submucous fibrosis and 50 oral cancer patients. The values were compared with that of 50 normal controls. The circulating immune complexes and their immunoglobulin contents were found to be elevated significantly both in oral submucous fibrosis and oral cancer. This study seems to be of help in monitoring the malignant transformation of oral submucous fibrosis to oral cancer.

Relation of transmembrane potential to cell survival following hyperthermia in HeLa cells.

Hyperthermia induces cell death and the usual endpoint to study this is the ability of the cells to form colonies. Hyperthermia is also known to alter membrane characteristics, especially transmembrane potential and this has been correlated with duration and degree of heating. The aim of the present study was to see the correlation between changes in membrane potential and clonogenic ability of HeLa cells after heat treatment of 41-44 degrees C. Membrane potential was measured by the fluorescence polarization of the plasma membrane probe 3,3'-dipentyloxacarbocyanine by flow cytometry. Cell survival was assessed by colony formation assay. The fluorescence intensity increased and cell survival decreased with an increase in temperature. The fall in survival following heat treatment closely paralleled the increase in fluorescent intensity, especially heat treatments of 60 min or more. After 2 h of heating at 44 degrees C, the surviving fraction decreased to 1% and the fluorescence intensity increased to 154.84% of the unheated controls. This study suggests that measurement of membrane potential by flow cytometry may potentially be an alternative to colony forming assay for assessing cell survival. Since the results of membrane potential measurements are available immediately, this has implications for its potential use as a predictive assay of thermosensitivity.

Serial cytological assay of micronucleus induction: a new tool to predict human cancer radiosensitivity.

BACKGROUND AND PURPOSE: The micronucleus test, generally done in cultured tumour cells irradiated in vitro, has not gained wide acceptance in predicting human cancer radiosensitivity. The purpose of this study was to see if micronucleus assay by serial scrape smear cytology can predict oral cancer radiosensitivity. MATERIALS AND METHODS: Forty nine oral cancer patients given radiotherapy (60 Gy/25 fractions/5 weeks) form the study population. Serial scrape smears were taken from their tumours before treatment and after delivery of 2, 5, 8 and 12 fractions, stained by Giemsa and the number of micronucleated cells (MNC) noted. The patients were grouped to those who developed tumour recurrence ('Resistant') and those who did not ('Sensitive'), and the pattern of micronucleus induction compared. RESULTS: Both groups of tumours had MNC even before treatment, with statistically significant dose-related increase with radiotherapy. The sensitive group had a higher mean increase in MNC count than the resistant group (6.1 times and 3.6 times the pre-treatment value, respectively) and better correlation with dose (r = 0.54 vs. 0.43). The increase in MNC count occurred earlier in the resistant group than in the sensitive, the TMNC (time for the pre-treatment value to double) being 3.3 days and 7.6 days, respectively. Also, the resistant group showed a plateauing of the MNC count which the sensitive group lacked. CONCLUSIONS: The higher MNC induction in the sensitive tumours suggests the usefulness of the assay as a test of radiosensitivity. The differing patterns of MNC increase suggest that differences in proliferation rate is an important cause of tumour failure. Serial cytological assay of micronucleus induction can identify both radiosensitivity and proliferation characteristics of tumours, and thus may turn out to be a useful test of radiocurability.

Clinical implications of cytogenetic classification in adult acute lymphoblastic leukaemia patients.

Cytogenetic analysis performed on pretreated unstimulated, bone marrow/peripheral blood samples of 46 adult patients with acute lymphoblastic leukaemia (ALL) showed sufficient metaphases in 39 patients and insufficient metaphases in 7 patients. G-banded karyotype analysis of these 39 patients revealed non-random clonal chromosome abnormalities in 31 patients and apparently normal karyotypes in 8 patients. Numerical abnormalities involving chromosome trisomies and structural abnormalities involving different types of chromosomal translocations and deletions were encountered in varying percentages. These patients were grouped into various cytogenetic subsets on the basis of their karyotype pattern and followed-up to evaluate their prognosis. Patients with apparently normal karyotypes showed good prognosis and those with 6q- showed intermediate prognosis. But all other patients with hyperdiploid, pseudodiploid and hypodiploid karyotypes were associated with poor prognosis. Cytogenetic classification of ALL patients is thus of clinical importance, as it helps the early identification of clinically important prognostic groups.

Differential expression of jackfruit-lectin-specific glycoconjugates in metastatic adenocarcinoma and reactive mesothelial cells-a diagnostic aid in effusion cytology.

Distinguishing reactive mesothelial cells from adenocarcinoma cells in serous effusions on the basis of morphological criteria alone is often difficult. Interest has therefore been focused on identifying reliable methods to supplement the conventional cytological techniques. Plant lectins have been reported as diagnostic markers for malignant cells. We studied 51 aspirated samples of benign and malignant effusions using horseradish-peroxidase-conjugated jackfruit lectin. No significant difference was observed between the cells of pleural and peritoneal fluids. The reactively proliferated mesothelial cells of benign effusions showed a predominance of mild staining while moderate and intense staining was predominant in malignant effusions. Intense and irregular lectin binding was observed in macrophages irrespective of the cause of effusion. The lectin staining method therefore appears to have some clinical significance as an additional diagnostic aid for use in effusion cytology.

Correlation of lectin binding with lymph node metastasis in oral cancers.

Lymph node metastasis is an important factor that influences the treatment policy and prognosis of oral cancers. The cell membrane is known to play a role in the metastatic process. The aim of the present study was to evaluate the relationship of lectin binding frequency of oral cancer cells to their capacity to metastasize to the lymph node. Smears collected from tumours of 66 untreated patients were stained with Jackfruit lectin (JFL) conjugated with horseradish peroxidase (HRP), with diaminobenzidine dihydrochloride (DAB) as the visualant. The frequency of cells showing lectin binding was evaluated. The results showed that tumours with a high frequency of JFL binding cells had higher risk of lymph node metastasis (RR = 1.5). It was also found that a combined score integrating the percentage of lectin binding cells with known clinical parameters, like primary tumour size, local invasion and histological subgroup, had better utility than any of these individually in assessing risk of lymph node metastasis. The density of sugar residues on the cell surface may be of importance in determining the lymph node metastatic potential of oral cancers, and the present study suggests the need for further research in this area.

Total hemolytic complement (CH50) and its fractions (C3 and C4) in the sera of diabetic patients with periodontitis.

The total hemolytic complement activity (CH50) and its fractions C3 and C4 were determined in 50 patients with Type II or non-insulin dependent diabetes mellitus (NIDDM) and 50 nondiabetic patients with periodontitis. The values were compared with those of 50 age and sex matched controls. An elevation of CH50 was observed in both the diabetic and nondiabetic patients, compared to controls. The diabetic patients with periodontitis showed a significantly higher complement activity compared to nondiabetic patients with periodontitis. The C3 and C4 values were significantly elevated in both diabetic and nondiabetic patients when compared with the control group. The elevation being more pronounced in diabetic groups.

Effect of hyperthermia on transmembrane potential of HeLa cells--a flow cytometric analysis.

The effect of hyperthermia on transmembrane potential was studied in HeLa cells in vitro using a 3',3'-dipentyl oxacarbocyanine [Di-0-C5(3)], a lipophilic cation probe that equilibrates across the plasma membrane according to the transmembrane potential. Uptake of the fluorescent probe was measured by flow cytometry. The flourescent intensity (FI) increased with increase in temperature, and the increase was statistically significant when the duration of heat treatment was 30 minutes or more. At each temperature studied the depolarization was higher after longer duration of heat treatment (p value: 41 degrees C < 0.05; 42 degrees C < 0.005; 43 degrees C < 0.001 and 44 degrees C < 0.001, respectively). The lack of significant depolarization after shorter duration of heating, particularly at lower temperatures could be due to the repair of membrane damage that could have occurred in the holding interval between heating and measurement. The results suggest that depolarization of membrane potential, i.e. increase in the intracellular cation concentration, can be considered as an indicator of cell injury by hyperthermia and may be mechanistically related to cell death by heat treatment. The technique may be suitable for studying repair of damage after hyperthermia.

Role of levamisole immunotherapy as an adjuvant to radiotherapy in oral cancer--immune responses.

Investigations were carried out to assess the effect of levamisole immunotherapy as an adjuvant to radiotherapy on the immune response of patients with squamous cell carcinoma of the oral cavity. Parameters assessed were leukocyte migration inhibition, response to PPD and oral cancer extract (OCA), lymphocyte transformation to PHA, circulating antibodies to OCA and circulating immune complexes (CIC). Comparisons were made between groups receiving levamisole (L), those receiving placebo (P) and normal controls. The results of a thirty-month follow-up are presented. Radiotherapy resulted in a depression of cell-mediated functions, reduction in antibody titer also showed a gradual increase with time of follow-up. Levamisole, however, appeared to reduce the levels of CIC.

Chromosome abnormalities in squamous cell carcinoma of the human oral cavity.

Cytogenetic studies carried out in tissues of 75 patients with squamous cell carcinoma of the oral cavity gave satisfactory results in 12 cases. Remarkable variation characterized the modal chromosome numbers of these tumors, ranging from marked hypodiploidy to tetraploidy. Chromosomes which were lost belonged to group A whereas chromosomes which were gained belonged to groups C, D, E, F and G. Marker chromosomes were present in three cases. There was no correlation between the chromosome abnormalities observed and the clinical stages of the disease. The pattern of chromosome abnormalities ranging from marked hypodiploidy to tetraploidy observed in oral cancer tissues suggests an association of DNA oncogenic virus possibly Herpes simplex virus Type I (HSV-1) with oral cancer.

Lectin cytochemistry in the exfoliative cytology of uterine cervix.

A lectin was isolated from the seeds of jack fruit (Artocarpus integrifolia) and purified using a column of immobilized N-acetyl-D-galactosamine. This jack fruit lectin (JFL) was then conjugated to horse-radish peroxidase (HRP) type VI and used to study the cell surface carbohydrate profile of the cytological smears of the uterine cervix using diaminobenzidine as substrate. Cervical smears from 15 healthy individuals and 65 patients with dysplasia, carcinoma in situ and carcinoma of uterine cervix were used for the study. Normal cells showed weak binding in the membrane as well as cytoplasm, whereas carcinomatous cells showed strong binding towards JFL. Carcinoma in situ cells showed a binding pattern similar to that of carcinoma. Dysplastic cells showed difference in binding in mild, moderate and severe dysplasia. The intensity of binding increased with the severity of the dysplasia. The nature and intensity of binding of jack fruit lectin with cancer tissues suggest that this lectin may be of use as a diagnostic aid in exfoliative cytology.

Altered expression of jack fruit lectin specific glycoconjugates in benign and malignant human colorectum.

Lectins bind to terminal non reducing sugars of glycoconjugates in cell membranes and secretions. These glycoproteins can be very characteristic, both for the differentiation state of a tissue and for its grade of malignancy. The carbohydrate structures of cellular glycoconjugates in normal, adenoma, and carcinoma of human colorectum were analysed using HRP conjugated Jack Fruit Lectin (JFL). Fifty two rectal carcinomas, 18 adenomas and 25 normal rectal tissues were used for the study. Diaminobenzidine (DAB) was used as the visualant. Normal rectal tissues showed positive reaction with intense staining in more than 60% of the cells. Adenomas were seen to have moderate to intense staining with a range of 30 to 60% positive cells. The lowest levels of JFL staining were observed in invasive tumours. JFL binding was found to decrease with carcinomatous cells compared to adenomas and normal cells. A significant negative association was found between JFL binding and histologic abnormality (r = -0.85, P <0.0000). These results suggest that there are alterations in the carbohydrate structures of cellular glycoconjugates, which can be related to goblet cell differentiation, in normal, benign and malignant human colorectal tissue.

Jack fruit lectin binding pattern in the exfoliative cytology of bronchopulmonary neoplasia.

N-acetyl D- galactosamine specific jack fruit lectin conjugate was used for the cyto-chemical study of benign and neoplastic lesions of the respiratory tract using diaminobenzidine as substrate on exfoliated cells and tissue sections. Sputum samples from patients with benign respiratory tract lesions (40), squamous cell Carcinoma (20), adenocarcinoma (16), small cell anaplastic carcinoma (12) and large cell anaplastic carcinoma (5) were used for the study. The lectin binding was strong in squamous cell carcinoma, large cell anaplastic carcinoma and adenocarcinoma, but variations in the binding pattern were observed in different carcinoma. Squamous metaplastic cells manifested slightly increased binding to lectin as compared to bronchial and native squamous epithelial cells. The nature of binding was eventually similar in cytology and histology. The ready availability and ease of preparation in purified from makes the jack fruit lectin a potential cytochemical reagent for sputum cytology.

Jack fruit lectin binding pattern in benign and malignant lesions of the breast.

N-acetyl D-galactosamine specific lectin was isolated from Jack fruit (Artocarpus integrifolia) and conjugated to horse radish peroxidase type VI. The purified conjugate was used for the study of tissue binding properties on benign and malignant lesions of the breast using diaminobenzidine as substrate on dewaxed tissue sections. Forty mammary carcinomas, 10 cystic hyperplasias of the breast and 10 normal breast tissues were used for the study. Neoplastic cells showed increased affinity to the lectin. The lectin binding was focally strong in neoplastic cells compared to the normal as well as the hyperplastic tissues. The stroma of the cancer tissues showed an intense strong binding where elastosis was present. The use of the lectin as a histochemical reagent is discussed.

Lectin binding as a prognostic indicator in gestational trophoblastic diseases (GTD).

Gestational Trophoblastic Diseases (GTD) is a group of hyperproliferative conditions of the placenta. Very often these can be fatal or recurrent. Presently, no reliable marker is available apart from serum beta HCG levels to identify tumours with a higher aggressive nature, the reduction pattern of the serum beta HCG levels indicating persistence of the disease. This causes a delay of nearly 12-16 weeks in deciding on chemotherapy. In this study, the potential of Jack fruit lectin (JFL) binding as a quick and cheap method of assessing the aggressiveness of the disease immediately after evacuation was evaluated. A significantly higher intensity of lectin binding was noticed in GTD when compared to gestational age related normal placentae. Persisting tumour lesions generally showed intense, diffuse and granular lectin binding and showed significant cytological atypia. The lectin binding score showed close correlation with the regressing pattern of serum beta HCG but not with the initial levels of beta HCG, indirectly pointing to its potential in identifying lesions with high risk of persisting disease. Hence evaluation of the lectin binding characteristics of the lesion immediately after evacuation will be of help in following up these patients closely and planning therapy.