Ch. Varalakshmi

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.

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.

Interaction Between B.7 and CD28 Costimulatory Molecules is Essential for the Activation of Effector Function Mediating Spontaneous Tumour Regression

The spontaneous regression of a rat histiocytoma, AK‐5, is mediated by activated natural killer cells through antibody‐dependent cellular cytotoxicity. In addition to the Fc–FcR interaction between the target and the effector cells demonstrated previously, we show the participation of costimulatory molecules B7 and CD28 in the efficient killing of the tumour cell. Blockade of the costimulatory interaction in vivo using anti‐CD28 led to increased tumour growth and a suppressed cytokine response. Anti‐CD28 antibody administration in vivo also diminished the cytotoxic potential of NK cells against AK‐5 cells in vitro. Our studies also demonstrate the expression of B7.1 and B7.2 antigen on AK‐5 tumour cells. The cytotoxic activity of natural killer cells was significantly inhibited when the effector/target cells were cultured in the presence of antibodies raised against B7.1, B7.2 and CD28. Administration of anti‐CD28 in vivo also affected the efficiency of the formation of effector/target conjugates in vitro. Similarly, anti‐CD28 injections affected expression of the adhesion molecules LFA 1 and ICAM 1 by splenocytes. Administration of anti‐B7.1 and B7.2 antibodies in AK‐5 tumour‐bearing animals showed a differential response. The cytotoxicity of natural killer cells was significantly inhibited after anti‐B7.2 administration, suggesting the preferential participation of B7.2 molecules in vivo. These observations suggest an important role for B7–CD28 interaction in AK‐5 tumour regression.

High intratumoural level of cytokines mediates efficient regression of a rat histiocytoma.

We have studied the role of cytokines in the spontaneous regression of AK‐5 histiocytoma in syngeneic rats. Animals in which the tumour regresses show high levels of cytokines in the serum compared with animals which succumb to the tumour, and levels of IL‐2, IL‐4, IL‐12, interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) are significantly higher in tumour tissue of the former. Thus there is an association between rejection of the tumour and the levels of cytokines present in the tumour mass. Our results also suggest a predominant Th1‐type of response in those rats that display early tumour rejection.

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.

Role of IFN-gamma produced after intraperitoneal transplantation of AK-5 cells in the induction of Fas ligand expression by tumor cells leading to immune evasion.

AK-5 tumor cells expressed Fas-L on their surface after intraperitoneal transplantation in syngeneic animals. Fas-L expression by AK-5 cells is involved in the killing of the effector cells. Thus, the tumor has developed an escape mechanism from immune attack. In the present study, we showed that Fas-L expression on AK-5 cells is regulated by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), as injection of antibodies against IFN-gamma downregulated the expression of Fas-L by tumor cells as determined by immunostaining and Northern hybridizations. Fas-L present on the tumor cells is biologically functional, as it induced DNA fragmentation in Fas+ YAC-1 cells. We have also shown shedding of Fas-L in cell-free ascitic fluid from tumor-bearing animals. These observations suggest that such cytokines as IFN-gamma and TNF-alpha play an important role in regulating the expression of Fas-L by AK-5 cells.

Development and characterization of a novel monoclonal antibody that recognizes an epitope in the central protein interaction domain of RapGEF1 (C3G)

The ubiquitously expressed protein RapGEF1 (C3G) regulates multiple cellular activities and is essential for early embryonic development in mammals. It has functions dependent on its catalytic activity as well as protein interaction domain and regulates β-catenin signaling. This study describes the generation of a novel monoclonal antibody, 3F6mAb and its characterization for recognition of RapGEF1. Mice were immunized with recombinant protein having only the Crk binding region of RapGEF1 and hybridoma clones created by fusion of immunized spleen cells with Sp2/0 myeloma cells. This antibody recognizes human, primate and murine RapGEF1 protein. Based on the recognition of various deletion constructs, we have mapped its epitope to 580–648 amino acids. Isotyping showed that it belongs to IgG1 class of heavy chain and Kappa light chain. 3F6mAb is suitable for detecting cellular RapGEF1 by western-blotting, immunofluorescence and immunoprecipitation. It has an advantage over most of the commercially available antibodies as it can detect N- and C-terminal truncated forms of RapGEF1. Using this antibody to detect mobility shift, we show that RapGEF1 is phosphorylated on tyrosine as well as S/T residues in its Crk binding domain. This monoclonal antibody is a valuable tool that will aid in understanding functions of cellular RapGEF1.