Putul Maity

Induction of colorectal cancer in rats by 20-methylcholanthrene.

Colorectal carcinoma was induced in Sprague-Dawley rats by 20-methylcholanthrene. Macroscopical studies revealed that the tumors, either sessile type or semi-pedunculated polyp, were generally observed after 32 weeks of the carcinogen treatment. In the distal colon 46.9% tumors appeared, whereas 20.4% and 32.6% tumors were found in the rectum and proximal colon, respectively. Sequential histopathological studies indicated that hyperplasia of goblet cells was common in early stages, which was reduced thereafter. Carcinogenesis progressed with the appearance of the different grades of dysplasia in colorectal mucosa with first incidence of the severe dysplasia in rats at the 20th week and in situ carcinoma at the close of 28th week. Most of the carcinomas were multifocal in origin and were well differentiated adenocarcinoma with primary invasion at the submucosa. In immunohistological studies, this carcinoma was also reactive with monoclonal antibody 660, prepared against a colorectal carcinoma associated mucin antigen.

Isolation and purification of vascular endothelial growth factor (VEGF) from ascitic fluid of ovarian cancer patients.

Vascular Endothelial Growth Factor (VEGF) or Vascular Permeability Factor (VPF) is an angiogenic cytokine expressed by many human and animal tumors. Because of the importance of VEGF in animal tumors, we purified VEGF/VPF from ascitic fluid of ovarian cancer patients with heparin sepharose column. The purified protein gave protein bands of 37 and 26 kD, respectively in 12% SDS PAGE. The specificity of the purified protein was determined with dot blot, trans-immunoblot and ELISA using polyclonal goat anti-VEGF antibody (Santa Cruz Biotechnology). The vasodilatatory effect of the purified protein was confirmed by a vascular permeability assay on mouse. A polyclonal mouse antibody was raised against the purified protein, which recognized the same protein by ELISA, transimmunoblot and dot-blot analysis. It has been also found that the raised polyclonal antibody in mouse- and the commercial VEGF polyclonal antibody (Santa Cruz Biotechnology) both inhibited in vitrocell proliferation of human MCF-7 cell line. This study shows for the first time an effort to purify VEGF from human source.(Pathology Oncology Research Vol 10, No 2, 104–108)

Angiogenesis : a putative new approach in glutamine related therapy

Angiogenesis or the generation of new blood vessels, is an important factor regarding the growth of a tumor. Hence, it becomes a necessary parameter of any kind in therapeutic studies. Glutamine is an essential nutrient of tumor tissue and glutamine related therapy involves clearance of circulatory glutamine by glutaminase. So, whether this enzyme has any effect on angiogenesis of a tumor or not becomes an obvious question. To address this question, this study has been carried out with different murine tumor models.The results indicate that purified glutaminase reduces tumor volume as well as restricts the generation of new blood vessels. Glutaminase is effective in the case of solid as well as ascites tumor models. In the case of induced cancer, the host exhibits delayed onset of neoplasia following enzyme treatment and tumor host interactions determine the intensity of the neovascularisation process. Therefore, it can be concluded that this enzyme might be an effective agent against cancer metastasis.

Antitumour activities of copper-ATP complex on transplantable murine lymphoma.

A synthetic metal-nucleotide complex [Cu3(ATP)26H2O]2- has been found to have a significant tumour inhibitory effect on Daltons lymphoma. Moreover, the haematopoietic system of the treated hosts is favourably disposed and the life span is much increased. It is noteworthy that the tumour inhibitory effect is enhanced by combination therapy.

Localization of phosphate dependent glutaminase in ascites fluid of ovarian cancer patient.

Phosphate dependent glutaminase was purified from ascites fluid of ovarian cancer patients. The purified enzyme showed a final specific activity of 110 unit / mg protein with 72 fold purification and 21% yield. Purified enzyme gives one dark band of Mr ~65.5 KD and two light bands of Mr ~47.5 KD and ~45 KD respectively on 10% SDS-PAGE. One major immunoreactive band was found in transimmunoblot analysis using antibodies against rat kidney and ascites fluid glutaminase raised in rabbit and mice respectively. Phosphate dependent glutaminase enzyme purified from mitochondria of malignant and non malignant ovarian tissue also showed bands of same molecular weight on 10% SDS-PAGE and gave same immunoreactive bands in trans-immunoblot like the purified glutaminase from ascites fluid. This result was confirmed by using the specific activity stain for glutaminase, which indicates that same enzyme activity is probably due to leakage of the same enzyme from malignant tissue into the ascites fluid. The purified enzyme from human peritoneal fluid showed a high specificity toward glutamine, therefore is a true glutaminase. Moreover, ascites fluid taken from patients of different age group with different stages of ovarian carcinoma revealed the presence of same glutaminase on 10% SDS-PAGE, and exhibited immunoreaction on ELISA, trans-immunoblot and dot immunoblot analysis.

Investigation on phosphate dependent glutaminase (EC 3.5.1.2) activity in host tissues of EAC-bearing mice and response of liver EC 3.5.1.2 on Cu-ATP therapy

Phosphate-dependent glutaminase activity in spleen, kidney, brain and liver is increased after tumor cell inoculation and this activity gradually increases with the progression of the tumor. The increase in enzyme activity in the liver is significant. Studies of the response of liver glutaminase after Cu-ATP treatment reveals that Cu-ATP is capable of reducing the high glutaminase level in subjects with malignant tumors to the normal level.

Synthesis, characterization, and in vitro cytotoxic effects of K4 [PtCl2ATP]

An antineoplastic agent, cis-K4 [PtCl2ATP] has been synthesized and characterized, using elemental analysis, solution conductance, thermoanalysis, infrared, NMR spectroscopy, and circular dichroism studies. The in vitro cytotoxic effect imparted by this compound on Daltons Lymphoma cells has been assessed by Trypan blue dye exclusion method and 51Cr release assays.

Investigation on glutamine amidohydrolase (EC 3.5.1.2) and glutamine aminotransferase (EC 2.5.1.15) activity in liver and plasma of EAC-bearing mice following glutaminase therapy

The anti-neoplastic activity of bacterial glutaminase on Ehrlich ascites tumor-bearing mice was studied by determining the reduction in the tumor cell count and extension of life span of the host after therapy. The therapeutic effect of glutaminase in relation to change in activity of glutaminolytic enzymes (glutamine amidohydrolase (GNase) and glutamine aminotransferase (GAt)) in liver and plasma were also studied. Bacterial glutaminase was shown to be effective in lowering the tumor burden with increased life span of the host. Glutamine amidohydrolase activity in the liver and plasma was raised significantly with increased tumor burden, whereas GAt activity remained unchanged. Following glutaminase therapy, this high level of GNase activity decreased in comparison to the untreated control. These changes were not seen when normal mice were treated with the same enzyme. Thus alteration in the enzyme levels, particularly GNase was observed to have some correlation with progression of the tumor growth.

A comparative study on expression of mucin related antigens in preneoplastic and neoplastic rat colorectal mucosa

A polyclonal antibody (PAb35) defined antigen (Ag) was characterized in association with two monoclonal antibody (MAbM1 and MAb660) defined mucin M1 and 660 Ags in colorectal mucosa during 20-methylcholanthrene-induced rat carcinogenesis in the same organ. Immunohistochemistry and ELISA revealed that these three antibodies were reactive with most of the colorectal carcinomas. MAbM1 and MAb660 were reactive with preneoplastic colorectal mucosa in rats with no detectable carcinoma, in contrast to non-reactivity with PAb35. PAb35 reacted with preneoplastic mucosa, present adjacent to the cancerous tissues only. Staining was mainly localized in the cytoplasm of cancer cells, goblet cells and luminal mucous deposits. In control rats, M1 and 660 Ags were present in gastric mucosa, but not in colorectum. PAb35 defined Ag was absent in gastrointestinal mucosa of controls. ELISA revealed 82% reduction in reactivity, when PAb35 was reacted with 2-mercaptoethanol (2ME) treated colorectal mucosal extracts. Ninety percent reduction was seen in the case of MAbM1. However, 50% reduction was demonstrated when MAb660 was reacted with 2ME treated extracts.

Anti-tumor efficacy of glutaminase-copper-ATP combination in mice bearing Ehrlich ascites carcinoma.

Glutaminase is a hematotoxic anti-tumor agent, and copper-ATP complex (Cu-ATP) is both anti-neoplastic and hematostimulatory. Combination chemotherapy with these two agents has been performed in mice bearing Ehrlich ascites carcinoma, to elucidate whether this could result in augmented tumor inhibition with reduced hematotoxicity. Glutaminase-Cu-ATP combination (glutaminase 250 IU/kg per day intraperitoneally for 10 days and Cu-ATP 2.5 mg/kg per day intraperitoneally for 10 days) was observed to be more effective in inhibiting tumor growth and in increasing the life span of the tumor hosts, compared with the individual efficacies of these two agents. Moreover, addition of Cu-ATP successfully prevented the hematotoxic effects of glutaminase in normal and in tumor-bearing animals. Thus glutaminase in combination with Cu-ATP holds promise for an effective cancer chemotherapeutic regimen.