Anti-tumour effect of humoral and cellular immunities mediated by a bacterial immunopotentiator,Lactobacillus casei,in mice

Summary Administration of a mixture containing Lactobacillus casei YIT 9018 (LC9018) and methylcholanthrene-induced fibrosarcoma (Meth A) cells into the peritoneum of syngeneic BALB/c mice suppressed the tumour growth and protected the mice from tumour death. With the appearance of the anti-tumour activity, serum complement-dependent tumour cytotoxic (CDC) antibody was induced on the 5th day after the administration as a result of the adjuvant effect. The cytotoxic antibody was not found in serum on the 5th day after inoculation of Meth A cells alone, but it was induced before the mice died of the tumours. Adjuvant induction of the cytotoxic serum antibody at an early time was also observed using Kirsten murine sarcoma virus-transformed tumour (K234) cells. Both of these cytotoxic antibodies in sera from Meth A-suppressed and the tumour-bearing mice were specific for the tumour cells and were IgM class, since they were absorbed with rabbit anti-mouse IgM antibody. However, the cytotoxic antibody was not found in the peritoneal cavity which was the tumour inoculation site, but binding antibody against the tumour cells was faintly detected in the region using an enzyme-linked immunoabsorbent assay (ELISA). In neutralization tests, the cytotoxic antibody did not exert anti-tumour activity in recipient mice when it was administered to the mice along with the tumour cells or when it was administered i. v. at the time of tumour inoculation. Moreover, the cytotoxic antibody was not available for the antibody-dependent cell-mediated cytotoxicity (ADCC). These results suggest that the cytotoxic antibody did not exert anti-tumour activity in the tumour-suppressed mice. In contrast, peritoneal exudate cells (PEC) on the 5th day, and PEC and spleen cells on the 15th day after i. p. administration of the mixture exerted strong anti-tumour activity as measured by the Winn test.In conclusion, the adjuvant effect of LC9018 induced tumour-specific humoral and cellular immunities but the anti-tumour activity was dependent only on the cellular effectors of the host. The possible use of LC9018 in tumour immunotherapy is discussed.     

Tumour-initiating cells vs. cancer 'stem' cells and CD133: what's in the name?

Recent evidence suggests that a subset of cells within a tumour have 'stem-like' characteristics. These tumour-initiating cells, distinct from non-malignant stem cells, show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumour cells, resistance to chemotherapy or radiation, and they are often characterised by elevated expression of the stem cell surface marker CD133. Understanding the molecular biology of the CD133(+) cancer cells is now essential for developing more effective cancer treatments. These may include drugs targeting organelles, such as mitochondria or lysosomes, using highly efficient and selective inducers of apoptosis. Alternatively, agents or treatment regimens that enhance sensitivity of these therapy-resistant "tumour stem cells" to the current or emerging anti-tumour drugs would be of interest as well.     

OP9-DL1 cell co-culture enhances anti-tumour immunity of mouse bone marrow-derived dendritic cells

DCs (dendritic cells) are the strongest professional APCs (antigen-presenting cells) to initiate immune responses against pathogens, but they are usually incompetent in initiating efficient immune responses in the progress of solid tumours. We have shown that Notch signalling plays a pivotal role in DC-dependent anti-tumour immunity. Compared with the control DCs, OP9-DL1 (Delta-like1) cell co-cultured DCs gained increased tumour suppression activity when inoculated together with tumour cells. This was probably due to the activation of Notch signalling in DCs enhancing their ability to evoke anti-tumour immune responses in solid tumours. Indeed, the OP9-DL1 cell co-cultured DCs expressed higher levels of MHC I, MHC II, CXCR4 (CXC chemokine receptor 4), CCR7 (CC chemokine receptor 7), IL-6 (interleukin 6), IL-12 and TNFα (tumour necrosis factor α), and a lower level of IL-10 than control DCs, resulting in more efficient DC migration and T-cell activation in vivo and in vitro. T-cells stimulated by OP9-DL1 cells co-cultured DCs more efficiently; and were cytotoxic against tumour cells, in contrast with control DCs. These results indicated that up-regulation of Notch signalling in DCs by co-culturing with OP9-DL1 cells enhances DC-dependent anti-tumour immune reactions, making the Notch signalling pathway a target for the establishment of the DC-based anti-tumour immunotherapies.     

Comparison of the rapid pro-apoptotic effect of trans-ß-nitrostyrenes with delayed apoptosis induced by the standard agent 5-fluorouracil in colon cancer cells

Trans-beta-nitrostyrene (TBNS) has been reported to be a potent inhibitor of protein phosphatases PTB1 and PP2A and to display a pro-apoptotic effect even in multidrug resistant tumour cells. Here we compared the anti-tumour potential of TBNS with 5-fluorouracil (5-FU) as the standard chemotherapeutic agent for colorectal cancer in LoVo cells. Resistance to 5-FU based therapy might be a consequence of 5-FU's delayed effect requiring long-term effective concentrations in the tumour tissue. Thus, alternatives like platin containing drugs with a more rapid effect have been introduced recently. Compared to 5-FU TBNS displayed a faster cytotoxic and pro-apoptotic effect. A 50% decrease in viability was observed already after 8 h with TBNS while 5-FU displayed no significant effect before 48 h. DNA fragmentation and caspase-3 assays confirmed the more rapid apoptotic effect of TBNS. Since apoptosis affects individual cells these results about a rapidly induced apoptosis were further studied on a single cell level in microscopic assays of caspase-3 and caspase-8 activation. Adducts of trans-beta-nitrostyrene displayed an anti-tumour effect comparable to TBNS which suggests the possibility of creating adducts with optimised tissue targeting. Finally, the calculation of a drug combination index displayed a synergistic effect for the combination of TBNS and 5-FU in Lovo as well as in HT-29 and HCT116 colon cancer cells.     

Nano-biotechnology in tumour and cancerous disease: A perspective review

In recent years, drug manufacturers and researchers have begun to consider the nanobiotechnology approach to improve the drug delivery system for tumour and cancer diseases. In this article, we review current strategies to improve tumour and cancer drug delivery, which mainly focuses on sustaining biocompatibility, biodistribution, and active targeting. The conventional therapy using cornerstone drugs such as fludarabine, cisplatin etoposide, and paclitaxel has its own challenges especially not being able to discriminate between tumour versus normal cells which eventually led to toxicity and side effects in the patients. In contrast to the conventional approach, nanoparticle-based drug delivery provides target-specific delivery and controlled release of the drug, which provides a better therapeutic window for treatment options by focusing on the eradication of diseased cells via active targeting and sparing normal cells via passive targeting. Additionally, treatment of tumours associated with the brain is hampered by the impermeability of the blood–brain barriers to the drugs, which eventually led to poor survival in the patients. Nanoparticle-based therapy offers superior delivery of drugs to the target by breaching the blood–brain barriers. Herein, we provide an overview of the properties of nanoparticles that are crucial for nanotechnology applications. We address the potential future applications of nanobiotechnology targeting specific or desired areas. In particular, the use of nanomaterials, biostructures, and drug delivery methods for the targeted treatment of tumours and cancer are explored.     

The proliferation rate paradox in antimitotic chemotherapy

Cytotoxic cancer chemotherapy drugs are believed to gain selectivity by targeting cells that proliferate rapidly. However, the proliferation rate is low in many chemosensitive human cancers, and it is not clear how a drug that only kills dividing cells could promote tumor regression. Four potential solutions to this "proliferation rate paradox" are discussed for the microtubule-stabilizing drug paclitaxel: drug retention in tumors, killing of quiescent cells, targeting of noncancer cells in the tumor, and bystander effects. Testing these potential mechanisms of drug action will facilitate rational improvement of antimitotic chemotherapy and perhaps cytotoxic chemotherapy more generally.     

Liposomes targeting tumour stromal cells

Liposomes have found clinical application in cancer therapy in the delivery of cytostatic agents. As a result of the targeted delivery of these toxic molecules to the tumour cells coupled to avoidance of toxicity-sensitive tissues, the therapeutic window is widened. Over the past years the focus of cancer therapy has shifted towards the stromal cells that are present in the tumour. It appears that clinically relevant tumours have acquired the ability to modulate the microenvironment in such a way that a chronic pro-inflammatory and pro-angiogenic state is achieved that contributes to invasion and metastasis and continued proliferation. Over the past years, liposomal formulations have been designed that target key stromal cell types that contribute to tumour growth. At the same time, many promising cell types have not been targeted yet and most of the studies employ drugs that aim at depleting stromal cells rather than modulating their activity towards an anti-tumour phenotype. In this review these target cell types will be addressed. Complementing these targeted formulations with the appropriate drugs to optimally suppress tumour-promoting signals while preserving anti-tumour action will be the challenge for the future.     

Genetic engineering, expression, and activity of a chimeric monoclonal antibody-avidin fusion protein for receptor-mediated delivery of biotinylated drugs in humans

The genetic engineering, expression, and validation of a fusion protein of avidin (AV) and a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR) is described. The 15 kDa avidin monomer was fused to the carboxyl terminus of the heavy chain of the HIRMAb. The fusion protein heavy chain reacted with antibodies specific for human IgG and avidin, and had the same affinity for binding to the HIR extracellular domain as the original chimeric HIRMAb. The fusion protein qualitatively bound biotinylated ligands, but was secreted fully saturated with biotin by COS cells, owing to the high level of biotin in tissue culture medium. Chinese hamster ovary (CHO) cells were permanently transfected with a tandem vector expressing the fusion protein genes, and high expressing cell lines were isolated by methotrexate amplification and dilutional cloning. The product expressed by CHO cells had high binding to the HIR, and migrated as a homogeneous species in size exclusion HPLC and native polyacrylamide gel electrophoresis. The CHO cells were adapted to a 4 week culture in biotin depleted medium, and the HIRMAb-AV fusion protein expressed under these conditions had 1 unoccupied biotin binding site per molecule, based on a [3H]-biotin ultrafiltration assay. The HIRMAb-AV increased biotin uptake by human cells >15-fold, and mediated the endocytosis of fluorescein-biotin, as demonstrated by confocal microscopy. In summary, the HIRMAb-AV fusion protein is a new drug targeting system for humans that can be adapted to monobiotinylated drugs or nucleic acids.     

E phage gene transfection associated to chemotherapeutic agents increases apoptosis in lung and colon cancer cells

The limited ability of conventional therapies to achieve the long-term survival of metastatic lung and colon cancer patients suggests the need for new treatment options. In this respect, genes encoding cytotoxic proteins have been proposed as a new strategy to enhance the activity of drugs, and combined therapies involving such genes and classical antitumoral drugs have been studied intensively. The E gene from phiX174 encodes a membrane protein with a toxic domain that leads to a decrease in tumour cell growth rates. Therefore, in order to improve the anti-tumour effects of currently used chemotherapeutic drugs on cancer cells, we investigated the association of the E suicide gene with these antineoplastic drugs. The E gene has antitumoral effects in both lung and colon cancer cells. In addition, expression of this gene induces ultrastructural changes in lung cancer transfected cells (A-549), although the significance of these changes remains unknown. The effect of combined therapy (gene and cytotoxic therapy) enhances the inhibition of tumour cell proliferation in comparison to single treatments. Indeed, our in vitro results indicate that an experimental therapeutic strategy based on this combination of E gene therapy and cytotoxic drugs may result in a new treatment strategy for patients with advanced lung and colon cancer.     

Liposomes targeting tumour stromal cells

Abstract

Liposomes have found clinical application in cancer therapy in the delivery of cytostatic agents. As a result of the targeted delivery of these toxic molecules to the tumour cells coupled to avoidance of toxicity-sensitive tissues, the therapeutic window is widened. Over the past years the focus of cancer therapy has shifted towards the stromal cells that are present in the tumour. It appears that clinically relevant tumours have acquired the ability to modulate the microenvironment in such a way that a chronic pro-inflammatory and pro-angiogenic state is achieved that contributes to invasion and metastasis and continued proliferation. Over the past years, liposomal formulations have been designed that target key stromal cell types that contribute to tumour growth. At the same time, many promising cell types have not been targeted yet and most of the studies employ drugs that aim at depleting stromal cells rather than modulating their activity towards an anti-tumour phenotype. In this review these target cell types will be addressed. Complementing these targeted formulations with the appropriate drugs to optimally suppress tumour-promoting signals while preserving anti-tumour action will be the challenge for the future.     

TQFL12, a novel synthetic derivative of TQ,inhibits triple-negative breast cancer metastasis and invasion through activating AMPK/ACC pathway

Thymoquinone (TQ) has been reported as an anti-tumour drug widely studied in various tumours, and its mechanism and effect of which has become a focus of current research. However, previous studies from our laboratory and other groups found that TQ showed weak anti-tumour effects in many cancer cell lines and animal models. Therefore, it is necessary to modify and optimize the structure of TQ to obtain new chemical entities with high efficiency and low toxicity as candidates for development of new drugs in treating cancer. Therefore, we designed and synthesized several TQ derivatives. Systematic analysis, including in vitro and in vivo, was conducted on a panel of triple-negative breast cancer (TNBC) cells and mouse model to demonstrate whether TQFL12, a new TQ derivative, is more efficient than TQ. We found that the anti-proliferative effect of TQFL12 against TNBC cells is significantly stronger than TQ. We also demonstrated TQFL12 affects different aspects in breast cancer development including cell proliferation, migration, invasion and apoptosis. Moreover, TQFL12 inhibited tumour growth and metastasis in cancer cell–derived xenograft mouse model, with less toxicity compared with TQ. Finally, mechanism research indicated that TQFL12 increased AMPK/ACC activity by stabilizing AMPKα, while molecular docking supported the direct interaction between TQFL12 and AMPKα. Taken together, our findings suggest that TQFL12, as a novel chemical entity, possesses a better inhibitory effect on TNBC cells and less toxicity in both in vitro and in vivo studies. As such, TQFL12 could serve as a potential therapeutic agent for breast cancer.     

TT-232: An Anti-tumour and Anti-inflammatory Peptide Therapeutic in Clinical Development

TT-232 is a structural derivative of the peptide hormone somatostatin with selective anti-proliferative and anti-inflammatory properties. It has a strong anti-tumour activity both in vitro and in vivo on a wide range of tumour models and induces apoptosis. Its anti-tumour activity is mediated through the SSTR1 receptor and by the tumour specific isoform of pyruvate kinase. TT-232 has been shown to be a potent neurogenic inflammation inhibitory, anti-inflammatory and analgesic agent with a broader spectrum than presently available anti-inflammatory/analgesic drugs. In animal models it is effective against neurogenic inflammation and blocks neuropathic hyperalgesia where COX-1 or COX-2 inhibitors (e.g. diclofenac or meloxicam) proved ineffective. TT-232 has passed phase I clinical trials without toxicity and significant side effects. Human phase II efficacy studies are ongoing in melanoma indication. Two more oncological indications and phase II clinical trials in inflammatory diseases, including rheumatoid arthritis and burn injuries are in preparation. This compound has the perspective to become the first drug in molecularly targeted therapy of inflammation where a combined effect of anti-inflammatory, analgesic and neurogenic inflammation inhibiting activity can be achieved.     

Tamoxifen and trifluoroperazine (Stelazine) potentiate cytostatic/cytotoxic effects of P-30 protein, a novel protein possessing anti-tumor activity

P-30 protein, a novel protein isolated in our laboratory from fertilized Rana pipiens eggs, has been shown to possess significant anti-proliferative and cytotoxic activity against a variety of human tumour cell lines. This protein also shows a potent anti-tumour activity in vivo in animal tumour models and is currently undergoing Phase I human clinical trials in cancer patient volunteers. The present study describes the in vitro effects of the concerted action of this protein and two other agents which affect the cell proliferative cycle. A significant potentiation of the P-30 protein-induced cell growth inhibition by tamoxifen as well as trifluoroperazine (Stelazine) in both the human A-549 lung carcinoma and the ASPC-1 pancreatic adenocarcinoma systems at wide ranges of drug concentrations was observed. The effect was apparently due to the synergistic action of P-30 protein and the agents tested. This data may provide clues that can be useful in explaining the mechanism of its anti-tumour activity. The results are also helpful for the designing in vivo animal and, perhaps eventually, human studies, whereby the combination therapies utilizing P-30 protein with agents of relatively low toxicity such as tamoxifen and/or Stelazine could offer a promising treatment(s) for these notoriously refractory types of human cancer.     

Tamoxifen and trifluoroperazine (Stelazine) potentiate cytostatic/cytotoxic effects of P-30 protein, a novel protein possessing anti-tumour activity

Abstract. P-30 protein, a novel protein isolated in our laboratory from fertilized Rana pipiens eggs, has been shown to possess significant anti-proliferative and cytotoxic activity against a variety of human tumour cell lines. This protein also shows a potent anti-tumour activity in vivo in animal tumour models and is currently undergoing Phase I human clinical trials in cancer patient volunteers. The present study describes the in vitro effects of the concerted action of this protein and two other agents which affect the cell proliferative cycle. A significant potentiation of the P-30 protein-induced cell growth inhibition by tamoxifen as well as trifluoroperazine (Stelazine) in both the human A-549 lung carcinoma and the ASPC-1 pancreatic adenocarcinoma systems at wide ranges of drug concentrations was observed. The effect was apparently due to the synergistic action of P-30 protein and the agents tested. This data may provide clues that can be useful in explaining the mechanism of its anti-tumour activity. The results are also helpful for the designing in vivo animal and, perhaps eventually, human studies, whereby the combination therapies utilizing P-30 protein with agents of relatively low toxicity such as tamoxifen and/or Stelazine could offer a promising treatment(s) for these notoriously refractory types of human cancer.     

Immunosenescence, suppression and tumour progression

There are good arguments for suggesting that two seminal papers published 50 years ago can be taken as the beginning of modern tumour immunology. These papers by R. Baldwin, “Immunity to transplanted tumour: the effect of tumour extracts on the growth of homologous tumours in rats” and “Immunity to methylcholanthrene-induced tumours in inbred rats following atrophy and regression of the implanted tumours” (Br J Cancer 9:646–51 and 652–657, 1955) showed that once tumours are established, they and their products can be recognised by the adaptive immune system and rejected. However, the tumour normally co-evolves with immunity, like a parasite, rather than being suddenly introduced as in these, and many other, experimental models. Dynamics of this co-evolution are illustrated by findings that inflammation enhances tumorigenicity, yet is important to enable T cells to respond properly to tumour antigen and exert anti-tumour effects. The important thing is to maintain the balance between effective anti-tumour immunity and tumour escape and/or stimulatory mechanisms. Tumours almost always co-exist with immune defence systems over extended periods and interact chronically with T cells. The effect of this is potentially similar to other situations of chronic antigenic stress, particularly lifelong persistent virus infection, most strikingly, CMV infection. The questions briefly explored in this symposium paper are what happens when T lymphocyte clones are chronically stimulated by antigen which is not or cannot be eliminated? What are the similarities and differences between chronic antigenic stimulation by tumour antigen versus CMV antigen? What can we learn in one system which may illuminate the other?     

Differential antioxidant/pro-oxidant activity of dimethoxycurcumin, a synthetic analogue of curcumin

Dimethoxycurcumin (Dimc), a metabolically stable analogue of curcumin, is under investigation as an anti-tumour agent. Recently a number of studies have been performed on Dimc in this laboratory and also by others. In the present article, all these results have been summarized and wherever possible compared with those of curcumin. Rate constant for reactions of Dimc with superoxide radicals was comparable with that of curcumin, while its reaction with peroxyl radicals was much slower. These results were further supported by the observations on the scavenging of basal ROS levels in lymphocytes and evaluation of antioxidant activities. In line with the earlier reports on curcumin, Dimc was a pro-oxidant and generated ROS in tumour cells. Both curcumin and Dimc were non-toxic to lymphocytes, while exhibiting comparable cytotoxicity to tumour cells. Additionally, these compounds showed higher uptake in tumour cells than in normal lymphocytes. Fluorescence studies on both the compounds revealed their binding to genomic DNA, similar sub-cellular distribution and nuclear localization. All these studies suggested that methylation of the phenolic-OH group in curcumin, although decreasing the antioxidant activity marginally, showed comparable pro-oxidant activity, making it a promising anti-tumour agent.     

In vivo effects of 5-fluorouracil and ftorafur[1-(tetrahydrofuran-2-yl)-5-fluorouracil] on murine mammary tumors and small intestine.

The in vivo anti-tumour and toxic effects of ftorafur (FT) and 5-fluorouracil (FU) were studied in the C3H mouse. On a molar basis, FU was two to three times more potent than FT with respect to growth inhibition of murine mammary adenocarcinomas. However, FT produced less host toxicity than FU when both drugs were compared at dose levels which produced equivalent anti-tumor effects. The differences between FT and FU with respect to tumor growth inhibition and host toxicity were reflected in their ability to suppress deoxyuridine incorporation into tumor cell and intestinal DNA, respectively. Flow cytometry (FCM) studies indicated that FT and FU were capable of producing pertubations in the DNA distribution of tumour cells. Both drugs induced an initial accumulation of cells in S phase following their administration at equivalent anti-tumour dose levels. At later intervals, an apparent block of cell progression at the G1/S boundary was observed. Drug-induced perturbations in the DNA distribution of tumour cells as detected by FCM correlated with results obtained by classical autoradiographic techniques using tritiated thymidine. Both procedures showed that tumor cells were capable of moving through S phase even in the presence of an apparently near complete inhibition of deoxyuridine incorporation into DNA. That such cells were, in fact, capable of synthesizing DNA at moderate rates was shown by their ability to incorporate 32P into DNA. The possible relationship of these findings to the therapeutic and toxic activities of FT and FU is discussed.