A. Mubarak Ali

Antitumor activity of curcumin is mediated through the induction of apoptosis in AK-5 tumor cells.

Curcumin, the yellow pigment of turmeric (Curcuma longa), used commonly as a spice, has been shown to possess anticarcinogenic activity. Curcumin inhibited AK‐5 tumor growth and induced apoptosis in AK‐5 cells. Curcumin induced apoptosis is mediated through the activation of caspase‐3, which is specifically inhibited by the tetrapeptide Ac‐DEVD‐CHO. In addition, curcumin induced tumor cell death is caused through the generation of reactive oxygen intermediates which is inhibited by N‐acetyl‐l‐cysteine. Our studies suggest that the apoptotic process induced by curcumin is the mechanism mediating AK‐5 tumor cell death.

Induction of stress response renders human tumor cell lines resistant to curcumin-mediated apoptosis: role of reactive oxygen intermediates

Abstract Curcumin, a well-known dietary pigment derived from Curcuma longa, has been shown to be a potent anti-inflammatory, antioxidant, and anticarcinogenic compound. The present study was designed to investigate the cytotoxic potential of curcumin against a range of human tumor cell lines in an attempt to understand its mechanism of action, which may lead to its possible therapeutic applications. We have shown that different cancer cell lines differ in their sensitivity to curcumin. Cell lines established from malignancies like leukemia, breast, colon, hepatocellular, and ovarian carcinomas underwent apoptosis in the presence of curcumin, whereas cell lines from lung, kidney, prostate, cervix, CNS malignancies, and melanomas showed resistance to the cytotoxic effects of curcumin. Sensitivity of the cancer cell lines to curcumin correlated with the generation of superoxide radicals as determined by the reduction of ferricytochrome C. Curcumin-resistant tumor cell lines showed significantly higher production of Hsp70, thus mounting a stress response and protecting the cells from the apoptotic cell death. These observations yield clues toward understanding the regulation of the cell death machinery by the stress proteins. Interestingly, curcumin had no effect on nontransformed cell lines, which showed neither superoxide generation nor the induction of a stress response. These observations demonstrate that curcumin is an interesting molecule with varied actions, depending on the cell type.

Immunomodulatory effects of curcumin : In-vivo

Curcumin specifically exhibits cytostatic and cytotoxic effects against tumors of multiple origin. Previously we have demonstrated apoptotic activity of curcumin against tumor cells with no effect on normal cells in-vitro. Many anti-cancer drugs exhibit deleterious effects on immune cells, which restrict their wide use in-vivo. In the present study, we have evaluated the effect of curcumin on the major functions of T cells, natural killer cells, macrophages and on total splenocytes in-vivo, which insight the role of curcumin on their broad effector functions. This study demonstrates that prolonged curcumin-injections (i.p.) do not impair the cytotoxic function of natural killer cells, the generation of reactive oxygen species and nitric oxide from macrophages and the levels of Th1 regulatory cytokines remained unaltered. Interestingly, curcumin-injections enhanced the mitogen and antigen induced proliferation potential of T cells. We have also evaluated immunomodulatory effects of curcumin in ascites-bearing animals. This study strengthens our belief that curcumin is a safe and useful immunomodulator for the immune system.

Differential cytotoxic effects ofAnnona squamosa seed extracts on human tumour cell lines: Role of reactive oxygen species and glutathione

Annonaceous acetogenins are a new class of compounds that have been reported to have potent pesticidal, parasiticidal, anti-microbial, cell growth inhibitory activities. In this study, organic and aqueous extracts from the defatted seeds ofAnnona squamosa (custard apple) were tested on different human tumour cell lines for antitumoural activity. While organic and aqueous extracts induced apoptosis in MCF-7 and K-562 cells, they failed to do so in COLO-205 cells. Treatment of MCF-7 and K-562 cells with organic and aqueous extracts resulted in nuclear condensation, DNA fragmentation, induction of reactive oxygen species (ROS) generation and reduced intracellular glutathione levels. In addition downregulation of Bcl-2 and PS externalization by Annexin-V staining suggested induction of apoptosis in MCF-7 and K-562 cells by both the extracts through oxidative stress. On the contrary, COLO-205 cells showed only PS externalization but no change in ROS and glutathione levels. These observations suggest that the induction of apoptosis byA. squamosa extracts can be selective for certain types of cancerous cells

Selective involvement of caspase-3 in ceramide induced apoptosis in AK-5 tumor cells

Ceramide, a product of sphingomyelin metabolism, is a novel lipid second messenger that mediates diverse cellular functions. The present study demonstrates the activation of caspase‐3/CPP‐32β, during apoptosis induced by cell permeable exogenous ceramides, in AK‐5 tumor, a spontaneously regressing rat histiocytoma. The apoptotic events were suppressed by the caspase‐3 specific tetrapeptide inhibitor DEVD‐CHO but not by the caspase‐1 inhibitor YVAD‐CMK. In cells overexpressing Bcl‐2, a significant decrease in cell death was observed after exogenous addition of ceramides. Furthermore the processing of caspase‐3 to its active form upon apoptotic stimulus, and the subsequent cleavage of the substrate PARP, suggested a central role for caspase‐3 in the ceramide mediated apoptosis in AK‐5 tumor cells.

Protection conferred by Bcl-2 expression involves reduced oxidative stress and increased glutathione production during hypothermia-induced apoptosis in AK-5 tumor cells

Hypothermia is known to retard mammalian cell growth, however, BC-8 cells, which have originated from AK-5 tumor after single cell cloning, were triggered into apoptotic pathway when grown at 30 degrees C. Cell death process showed typical apoptotic features like DNA fragmentation, cytochrome c release, etc. Introduction of Bcl-2 gene in BC-8 cells inhibited hypothermia-induced apoptotic process, which is ascribed to reduced ROS generation and higher glutathione production. Thus, Bcl-2 seems to control the apoptotic induction process at the level of redox regulation, in addition to its known effects at the mitochondrial dysregulation. These observations suggest that tumors, which are low in Bcl-2 expression, are sensitive to hypothermic shock and make hypothermia an interesting inducer of apoptosis in tumor cells.

Administration of anti-IL-12 antibody in vivo inhibits rejection of a rat histiocytoma and suppresses cytokine response in a tumour-bearing host

Activated macrophages are the major producers of heterodimeric cytokine interleukin‐12 (IL‐12). Earlier evidence suggested that early rejection of AK‐5 tumours is mediated by IL‐12 through interferon‐γ (IFN‐γ) production, involving activation of natural killer (NK) cells and upregulation of T‐helper 1 (Th1)‐type cytokine response. Injection of anti‐IL‐12 antibody into AK‐5 tumour‐bearing animals resulted in a large number of changes in the host immune response towards the tumour. These animals showed diminished NK‐mediated antibody‐dependent cell‐mediated cellular cytotoxicity (ADCC) activity, down‐regulation of Th1‐type cytokine response, decreased anti‐tumour antibody response ultimately leading to either delay or inhibition of the tumour‐regression process. There was also increased production of IL‐10 in the animals that had received anti‐IL‐12 antibody thereby resulting in the down‐regulation of IL‐2 and IFN‐γ production. IL‐12 plays a major role in the activation of the different immune parameters responsible for early rejection of AK‐5 tumour. We also studied the activation status of macrophages from tumour‐transplanted animals and their ability to produce IL‐12. Monocytes/macrophages from antibody‐injected animals were less active and produced lower quantities of IL‐12, IL‐1β and tumour necrosis factor‐α (TNF‐α) as compared with the macrophages from AK‐5 tumour‐bearing animals.

Apoptosis: the in vivo mechanism which mediates spontaneous AK-5 tumor regression

We have studied the mechanism of induction of apoptosis in a rat histiocytoma in vivo. Tumor cells are killed by necrosis and apoptosis when injected into immune animals; however, naive animals are incapable of killing the tumor cells. Tumor cells show DNA fragmentation within 3 h after transplantation in the peritoneal cavity. Natural killer (NK) cells act as the effector in causing tumor cell death through apoptosis since animals depleted of their NK cell population did not show target cell DNA fragmentation. Herbimycin A inhibits the induction of apoptosis, suggesting the requirement for protein phosphorylation.

Generation of cytotoxic monoclonals against AK-5 histiocytoma: conjugation with daunomycin and use in chemotherapy

We have successfully generated cytotoxic monoclonal antibodies against a rat histiocytoma, AK-5. Monoclonal antibodies obtained after fusing immunized rat splenocytes with SP2/0 myeloma, were cytotoxic to AK-5 cells in the presence of complement. These monoclonals were highly specific and did not show any cross reactivity with normal cells and ascitic tumors such as Zajdela ascites hepatoma or Meth A. One of the antibodies was conjugated to daunomycin and used in the chemotherapeutic treatment. Total regression of AK-5 histiocytoma was obtained after injection of daunomycin-MAb conjugate into tumor bearing animals suggesting the specific targeting of the antineoplastic drug to the tumor. The histology of the tumor sections showed extensive necrosis of the tumors after treatment of the animals with drug-MAb conjugate.

Serum protects HeLa cells from antiestrogen effects in culture.

The effect of nafoxidine and tamoxifen on HeLa cells is reported in this study. The two antiestrogens showed no effect on HeLa cells when grown in the presence of fetal calf serum, the cytotoxic effects were observed only under serum-free conditions. BSA also protected the HeLa cells from antiestrogen effects. The effect of nafoxidine and tamoxifen on different established cell lines and a primary culture of rat skin fibroblasts has been studied.