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.

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.

Antitumour Effects of Isocurcumenol Isolated from Curcuma zedoaria Rhizomes on Human and Murine Cancer Cells.

Curcuma zedoaria belonging to the family Zingiberaceae has been used in the traditional system of medicine in India and Southwest Asia in treating many human ailments and is found to possess many biological activities. The rationale of the present study was to isolate, identify, and characterize antitumour principles from the rhizomes of Curcuma zedoaria, to assess its cytotoxic effects on human and murine cancer cells, to determine its apoptosis inducing capacity in cancer cells, and to evaluate its tumour reducing properties in in vivo mice models. Isocurcumenol was characterized as the active compound by spectroscopy and was found to inhibit the proliferation of cancer cells without inducing significant toxicity to the normal cells. Fluorescent staining exhibited the morphological features of apoptosis in the compound-treated cancer cells. In vivo tumour reduction studies revealed that a dose of 35.7 mg/kg body weight significantly reduced the ascitic tumour in DLA-challenged mice and increased the lifespan with respect to untreated control mice.

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.

Significance of a galactose specific plant lectin for the differential diagnosis of adenocarcinoma cells in effusion.

BACKGROUND: Distinguishing adenocarcinoma cells from reactively proliferated mesothelial cells and macrophages is one of the greatest challenges in the cytodiagnosis of effusions. Aberrant glycosylation of cell surface glycoconjugates is emblematic to malignancy, and lectins being an important class of probes to demonstrate these aberrations, lectin cytochemistry is of great interest to differentiate adenocarcinoma cells from reactive mesothelial cells. AIM: The present study analyzed the potential of a plant lectin to distinguish malignant cells from reactive mesothelial cells and macrophages. MATERIALS AND METHODS: Snake gourd lectin (SGL) was isolated, purified and conjugated to horse radish peroxidase (HRP) and incubated with the cells of benign (46) as well as malignant (39) effusions using the standard immunocytochemical method with diaminobenzidine as the chromogen. The lectin-bound areas were quantitatively assessed as mild, moderate and intense binding. STATISTICAL ANALYSIS: The mean score for benign and malignant effusions were statistically analyzed. Student's 't'-test was performed to assess the significance. RESULTS: The lectin HRP complex bind to the cytoplasm of benign and malignant cells as well as macrophages. A significantly higher score for intense binding (P = 0.001) was found to differentiate malignant cells from reactive mesothelial cells. Macrophages showed intense irregular binding. CONCLUSIONS: SGL binding assay can play a role in the differential diagnosis of metastatic adenocarcinoma in effusions.

Radiation-induced changes in oral carcinoma cells - a multiparametric evaluation.

The aim of this study was to see whether serial cytological evaluation of various cellular abnormalities in tumours from patients receiving fractionated radiotherapy can predict radio-response in oral carcinoma. Cytological assessment was carried out in scrape smears collected prior to and during the course of radiotherapy in 68 patients with squamous cell carcinoma of the oral cavity planned for radical radiotherapy with accelerated fraction schedule. Smears were evaluated for a set of 15 radiation-induced cellular abnormalities. The relationship between the cellular alterations and the cumulative radiation dose was analysed by Kruskal-Wallis one-way anova. The results showed that among the various quantifiable changes that occur in irradiated cancer cells, karyolysis, karyorrhexis, pyknosis, cytolysis, multinucleation, micronucleation and nuclear budding show significant increase depending on the dose of radiation. The radio-resistant group of patients exhibited a lesser degree of change compared with the radio-sensitive group. This suggests that radio-resistance may be due to the defective induction of cell damage and that these cytological features may have potential use as predictive markers of radio-sensitivity in oral carcinoma.

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.

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.

Radiation-induced acute immediate nuclear abnormalities in oral cancer cells: serial cytologic evaluation.

OBJECTIVE: To evaluate the dose-response relationship of nuclear abnormalities in tumor cells collected by serial scrape smears from oral cancer patients on fractionated radiotherapy. STUDY DESIGN: The study included 31 patients with squamous cell carcinoma of the oral cavity treated by radiotherapy (60 Gy in 25 fractions; 2.4 Gy per fraction). Serial scrape smears were taken from each tumor before treatment and after delivery of various fractions, usually 2 (4.8 Gy), 5 (12.0 Gy), 8 (19.2 Gy) or 12 (28.8 Gy). The smears were stained by Giemsa stain and evaluated by light microscopy, and the number of micronucleated, binucleated, nuclear budded and multinucleated cells were scored. Their relation to cumulative dose was analyzed by Kruskal-Wallis one-way analysis of variance. The results were expressed in terms of 1,000 mononucleated cells. RESULTS: Even before treatment, most of the tumors showed various abnormally nucleated cells, and, despite the high intertumoral variation (as indicated by the high variance), all of them showed statistically significant dose-related increases. The mean values before treatment and after irradiation with 28.8 Gy, respectively, were 2.8 and 19.5 (P < .0001) for micronucleated cells, 1.5 and 8.5 (P < .000001) for nuclear budded cells, 8.2 and 35.5 (P < .0001) for binucleated cells, and 3.7 and 16.8 (P < .0001) for multinucleated cells. When the different types of nuclear abnormalities were combined and analyzed as "abnormally nucleated cells," the mean count before treatment and after 28.8 Gy were 7.9 and 44.9 (P < .00001), respectively. CONCLUSION: The study showed that radiation-induced micronucleation, multinucleation, binucleation and nuclear budding in oral cancer cells has statistically significant dose-related increases that become evident in the initial few days of radiotherapy and that they can be differentiated well by cytology. This dose-response relationship and the high intertumoral variations suggest that serial assay of these changes has potential use for radiosensitivity prediction.

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.

Expression of lectin binding in oral submucous fibrosis.

Lectins are a group of specific glycoproteins present in cells, particularly cell membrane. Recently, lectin binding studies have been used as a diagnostic as well as prognostic indicator of neoplasm's. Oral submucous fibrosis (OSMF) is a potential premalignant condition predominantly seen in Indian subcontinent. A comparison of expression of lectin binding was studied in normal tissue, OSMF cases and oral squamous cell carcinoma. The OSMF cases were grouped into early and advanced conditions as per the histopathologic criteria. Patterns of lectin binding observed with advanced OSMF cases were comparable with that of Oral squamous cell carcinoma. The role of lectin binding studies in assessing the malignant potential of a pre-malignant condition is discussed.

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.

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.

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.

Lectin histochemistry of oral premalignant and malignant lesions: correlation of JFL and PNA binding pattern with tumour progression.

The expression of glycoconjugates specific to Jack fruit lectin (JFL) and peanut agglutinin (PNA) in various clinicopathological stages of tumour progression in the oral mucosa were studied. These included various clinical forms of dysplastic and non-dysplastic oral leucoplakias, carcinomas, normal keratinising (gingiva) and non-keratinising (buccal mucosa) epithelia. It was seen that the binding patterns of PNA and JFL in the epithelial cells of various types of oral lesions were more or less similar. Normal non-keratinising epithelium showed mild membrane staining only in the spinal layers, while normal keratinising epithelium showed a moderate membrane staining and mild cytoplasmic staining in all layers. Moderate membrane and mild cytoplasmic staining were observed in leucoplakias, irrespective of various clinical or histological types. In carcinomas, the intensity of lectin binding was high, particularly in the membrane of differentiated cells. Correlation analysis of the binding pattern of PNA and JFL showed significant correlation in the membrane and cytoplasm of all layers with histological stages of tumour progression. The present study thus showed that PNA and JFL may be used as cytochemical probes in differentiating malignancy from benign lesions of the oral mucosa.

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.

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.

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).

Oral squamous cell carcinoma in a case of dyskeratosis congenita.

Oral manifestations of dyskeratosis congenita (DCG) have received little attention in dental literature. This report is a case of dyskeratosis congenita in a 17-year-old female which was associated with oral lesions such as leukoplakia, superimposed candidal infection, desquamative gingivitis, and severe periodontitis. Histopathologic examination of the granular lesion on the right lateral border of the dorsum of the tongue showed well-differentiated squamous cell carcinoma. Etiopathogenesis, clinical features, and laboratory findings of this disease are discussed.