Use of formaldehyde as a novel agent for cancer therapy

International Research Journal of Biotechnology (ISSN: 2141-5153) Vol. 2(5) pp.093-102, April, 2011 Available online http://www.interesjournals.org/IR...
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International Research Journal of Biotechnology (ISSN: 2141-5153) Vol. 2(5) pp.093-102, April, 2011 Available online http://www.interesjournals.org/IRJOB Copyright © 2011 International Research Journals

Full Length Research Paper

Use of formaldehyde as a novel agent for cancer therapy Prabir Chakravarty Tumor Immunity and Gene Therapy Unit, Chittaranjan Cancer Institute, S.P. Mukherjee Road, Calcutta-700 025, India. Accepted 11 April 2011

Formaldehyde is a ubiquitous aldehyde found in the environment. However, its biological role has not been fully understood. It is known to be extensively used in alternative forms of medicine for treating a wide range of diseases including proliferating disease like cancer. However, there is no study on its possible role in experimental cancer therapy. The present study was undertaken to explore the possible role of formaldehyde in cancer and its implications, if any in cancer therapy. Experiments were carried out in vitro and in vivo to investigate the probable role of formaldehyde in proliferation, cytotoxicity and in cancer therapeutics. The study revealed that formaldehyde at a lower concentration of 50ng/ml increased proliferation of tumor cells. However, when the concentration was enhanced, an inhibitory effect on tumor cell proliferation was noted. A formaldehyde concentration of 200ng/ml was cytotoxic to tumor cells in vitro. To ascertain its in vivo effectiveness for cancer therapeutics, tumor bearing animals were administered with different concentrations of formaldehyde and monitored; the end point being survival of the animals. It was demonstrated for the first time that formaldehyde at a concentration of 118.4µ µg/ Kg body weight had a therapeutic effect on tumor bearing animals. This concentration was found to be cytotoxic to tumor cells in vivo without causing any adverse effect on the physiology of animals, About 25% of treated tumor bearing animals survived for more than four months following tumor cell transplantation compared to untreated tumor bearing animals, all of which died within one month of tumor cell transplantation. In conclusion It appears that an appropriate concentration of formaldehyde has a therapeutic role without being cytotoxic to animals. This insight could open a new vista for treating cancer in future. Keywords: Formaldehyde, Cancer, Therapy, Cytotoxicity assay, Proliferation assay and Tumor

INTRODUCTION Formaldehyde is a ubiquitous colorless, flammable, strong-smelling chemical that is used in building materials and to produce many household products. In addition, formaldehyde is commonly used as an industrial fungicide, germicide, and disinfectant, and as a preservative in mortuaries and medical laboratories (NIOSH, 1967). It occurs naturally in the environment and is produced in small quantities by most living organisms as part of their normal metabolic processes. In humans and experimental animals, formaldehyde is

*Corresponding author Email: [email protected]

readily absorbed by all exposure routes. When inhaled, it reacts rapidly at the site of contact and is quickly metabolized in the respiratory tissue. Humans experience sensory irritation (eye, nose etc); though no systemic toxicity has been observed following repeated exposure to formaldehyde in animals and humans (NIOSH, 1976). Formaldehyde has long been known to have carcinogenic property with direct association with the etiology of some forms of cancer (Nordman et al, 1985). The possible relationship between formaldehyde exposure and cancer has been studied extensively in experimental animals and humans. There is clear evidence of nasal squamous cell carcinoma (SCC) from

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inhalation studies in the rat, but not in mouse and hamster. Although several epidemiological studies of occupational exposure to formaldehyde have indicated an increased risk of nasopharyngeal cancers, the data are not consistent. The postulated mode of action for nasal tumors in rats is biologically plausible and considered likely to be relevant to humans. Recent studies have suggested a possible link between exposure to formaldehyde and nasal cancer as was hypothesized based on epidemiological studies (Broughton and Thrasher, 1988). An NCI case-control study among funeral industry workers that characterized exposure to formaldehyde also found an association between increasing formaldehyde exposure and mortality from cancers of the hematopoietic and lymphatic systems, particularly myeloid leukemia (Collins and Lineker, 2004; Checkoway et al, 2010). However, formaldehyde is also known to have various advantageous effects and is widely used in different biological fields. It is used directly or in formulations in a number of industries including medicine-related industries (such as forensic/hospital mortuaries and pathology laboratories). It is known to have disinfective properties and is extensively used in tissue culture and in the synthesis of pharmaceutical drugs (Olsen and Dosing, 1982). Its role as tissue preservatives is also well documented (Grammer et al, 1990). It is important to note that formaldehyde does not have any adverse effects on normal physiology of an organism up to a dosage of 80mg/kg body weight (Thrasher et al, 1989). Formaldehyde is also known to have systemic effects when taken in vivo. The systemic changes include varied Immune changes with formaldehyde exposure. Studies have revealed changes in cell-mediated immunity like changes in basophiles and/or suppressor cells (Pross et al, 1987; Thraser et al, 1988; Thraser et al, 1990). It could also influence the immune response indirectly by increasing nitric oxide production through activation of the brain vanilloid receptor (Palazzo et al, 2002). Thus formaldehyde appears to be a versatile molecule whose potential beneficial effects have not yet been fully exploited. Though its importance has long been recognized in some forms of alternative medical practices like homeopathic medicine and as tissue preservatives, not much research or application have been done in main stream medicine. In homeopathic medicine, formaldehyde is extensively used in very low concentrations to treat various pathological conditions including proliferative disease like cancer in humans (Boericke, 2002). However, no exhaustive studies have been conducted as yet and experimental evidences are lacking to establish its usefulness for cancer therapy in humans. In this direction, we have reported earlier that formaldehyde have antiproliferative activity and may have some use in therapy of cancer (Chakravarty et al, 1995). We hypothesized that formaldehyde at certain

concentrations may be effective in treating proliferative diseases like cancer in vivo. In this study, extensive in vitro and in vivo experiments were carried out in an animal tumor model in an effort to ascertain its role if any, in cancer. Here we show for the first time that formaldehyde has both proliferative and anti-proliferative property at different concentrations and its antiproliferative activity could be successfully exploited to treat cancer in an experimental tumor model.

MATERIALS AND METHOD

Tumor Model Studies were carried out with transplanted Earlich’s Ascites Carcinoma (EAC) in vivo and in vitro. The cells were maintained in vivo in the swiss mice by serial passage. The animals received 1x107 tumor cells by ip injection in the abdomen. The tumor volume reached its maximum by day 17 and thereafter maintained a stagnant condition. The animals eventually died between 20-25 days following tumor transplantation.

In vitro studies In vitro studies were carried out to determine the effect of formaldehyde (Fisher, NJ) on tumor cells.

Proliferation assay For proliferation assay, the EAC cells were extracted, washed and suspended in RPMI 1640 media containing 5 10% FCS. 100µl containing 1x10 cells were seeded in each well of a 96 well plate. To each well freshly prepared formaldehyde solution at different concentrations were added for a final volume of 200 µl/well. For each concentration six wells were used. Appropriate controls were used against the formaldehyde treated cells. After 48 hours of incubation oneµci/µl of 3H thymidine was added to each well and the incorporation of 3H Thymidine by the EAC cells was determined in a scintillation counter.

Cytotoxicity assay For determining the cytotoxicity of formaldehyde on tumor cells, 1x106 cells were seeded in each well of a 24 well plate. The concentration used for the cytotoxicity assay was similar to that used in the in vivo study. Hundred micro liters (µl) of formaldehyde solution (8

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µg/ml) was added in a final volume of 0.5 ml. After 48 hours, viability of the cells was checked by trypan blue exclusion test. The results are expressed as percent • dead cells. At least 500 cells were counted for the • purpose • In vivo studies

Mice Six to 8 week-old male (swiss strain) mice was obtained from the Institutes animal Resources facilities and maintained under controlled temperature, humidity, and a 12h light: dark cycle with food and water ad libitum.

solution (a dose of 8µg/ml [118.4µg/ Kg body weight] in the following schedule: Group CI – For 10 consecutive days Group CII – For 15 consecutive days Group CIII – For 20 days; of which first 15 were administered on consecutive days and the remaining five were given at an interval of 2 days (a total of 25 days). A group of 15 animals served as controls which received 0.4ml of normal saline for 20 consecutive days. In order to determine the effect of the therapeutic dose on normal mice, 15 normal animals were injected with formaldehyde solution (118µg/kg body weight) for 20 consecutive days (Group E). The Group E animals were regularly weighed and blood picture (total RBC count, total WBC count, differential count was monitored weekly. All the treated and non-treated tumor animals were monitored for tumor growth.

Survival Studies

Statistics

Animals: Male, six to eight weeks old mice were used for the study. The tumor strain used was EAC maintained in ascetic form in the peritoneal cavity of the animals. About 300 hundred animals were used for the entire study. For experimentation, each animal was injected ip with viable 1x105 EAC cells in the peritoneal cavity. The viability of the tumor cells was determined by counting the tumor cells in an improved neubeur chamber by the trypan blue exclusion test. For establishing the effective therapeutic regimen of formaldehyde based on the information of in vitro studies, the animals were divided into four groups (group A, B, C and D). For each group 30 animals were used. Each animal from the respective groups and controls 5 were injected ip with viable 1x10 EAC cells in the peritoneal cavity. Twenty four hours later each of the animals from respective groups received the following concentration of formaldehyde solution ip in a final volume 0.4 ml: • Group A : 2µg/ml [29.6µg/Kg body weight ] • Group B ; 4µg/ml [59.2µg/ Kg body weight] • Group C: 8µg/ml [118.4µg/ Kg body weight] • Group D: 16µg/ml [236.8µg/Kg body weight] All the animals belonging to different groups were treated for 7 consecutive days with formaldehyde solution. The animals were monitored for 4 months and beyond to assess the therapeutic effect using survival as the end point. As the group C treated animals receiving a dose of 8µg/ml [118.4µg/ Kg body weight] showed increased survival following treatment with formaldehyde. Further modifications of the treatment regimen were deployed in an effort to increase the efficacy of the treatment. In this protocol, tumor bearing animals received formaldehyde

The statistical analysis was done by Student’s t-test. The results were considered significant when p < 0.05. The work was done at Tumor Immunity and GeneTherapy Unit, Chittaranjan National Cancer Institute, Calcutta- 700025, India. The animal use protocol (AUP) was duly approved by the Animal ethics committee of the Institute.

RESULTS Incorporation of 3H-Thymidine by tumor cells in presence of formaldehyde solution showed increased proliferation The result obtained in the proliferation assay is shown in figure 1. The figure 1 shows that there was an inverse correlation between tumor cell proliferation and formaldehyde concentration. While no proliferation of tumor cells was noted at a concentration of 200 ng/ml and at a concentration of 100 ng/ml, there was however, an increased proliferation in tumor cells, as reflected by 3 H-Thymidine incorporation, at a much lower concentration of 50 ng/ml. The increase was 17% more than that of the controls, suggesting an increased rate of proliferation of tumor cells in presence of formaldehyde. From this experiment, it was also ascertained that a concentration of 200ng/ml could be suitable for inhibiting tumor growth in vivo.

Formaldehyde is cytotoxic to tumor cells The result obtained from in vitro cytotoxicity assay is depicted in figure 2. It is evident from the figure that the

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Figure 1. Effect of formaldehyde on tumor cell proliferation The figure shows Incorporation of 3H-Thymidine by tumor cells in presence of formaldehyde solution. An increased proliferation was noted at lower concentration while inhibition at higher concentration.

Figure 2. Effect of formaldehyde on tumor cell viability The figure shows the viability of tumor cells in presence of formaldehyde. The figure shows that formaldehyde was cytotoxic to tumor cells. The results are shown as mean ± SEM. The level of significance when p< 0.05

cells treated with formaldehyde showed cytotoxicity at 48 hours of treatment. The percent of killed cells was as

high as 60% compared to controls. These tumor cells also exhibited necrotic features with ruptured membrane

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Figure 3. survival of mice in different experimental groups treated with formaldehyde The figure shows the survival of animals following treatment with formaldehyde at different concentrations: Group A : 29.6µg/Kg body weight; Group B ; 59.2µg/ Kg body weight; Group C: 118.4µg/ Kg body weight, Group D: 236.8µg/Kg body weight, respectively. The level of significance when p< 0.05.

while viewed under microscope. Survival study

Increased survival of animals following treatment with formaldehyde The figure 3 shows the survival of mice in different experimental groups treated with different concentration of formaldehyde. It is evident from the figure that group C mice injected with formaldehyde at a concentration of 118.4µg/ Kg body weight survived the maximum period of time following tumor cell transplantation without showing any signs of toxicity. However, with increase in 6 the number of tumor cells (1x10 ) there was no effect on survival and the animals died by three weeks of tumor cell transplantation as was the case of control animals injected with similar number of EAC cells. This confirms that tumor burden along with concentration of formaldehyde is an important criteria for tumor control in vivo. It is also to be noted that the animals in group A and group B receiving lower concentration of formaldehyde did not impact on survival rather they died in similar time span or earlier like the untreated animals. However, the animals receiving higher concentration of

formaldehyde (group D) did not impact on survival rather they died in similar time span or earlier like the untreated animals. This may be due to other responses generated by formaldehyde administration. The study therefore, showed that the used concentration of 8µg/ml or 118µg/kg body weight (Group C) was suitable for therapeutic use when the initial cell number injected was 100,000. To increase the efficacy of the treatment schedule on survival of tumor bearing animals, the dose of 8µg/ml or 118µg/kg body weight was then tried in different time schedule. The figure 4 shows the result obtained from the study. It is evident from the figure 4 that the tumor bearing animals that did not receive any treatment died by four weeks of tumor cell transplantation. The treated animals however, showed variable results. Some of the animals of group CI survived for more than 12 weeks after tumor cell transplantation. The earliest death due to tumor burden was not before fifth week of tumor cell transplantation. The other two groups (CII & CIII) had interesting results. While they both showed death due to tumor burden from eight weeks on wards; the rate slowed down gradually in group CIII mice and about 25 percent of the animals were surviving after 17 weeks of tumor cell transplantation. In group CIII, however five animals died without tumor burden at different time intervals following

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Figure 4. survival of animals following formaldehyde treatment with improved schedule (Group C) The figure shows the survival of animals following treatment with formaldehyde at the concentration of 118.4µg/ Kg body weight (Group C) with improved schedule as mentioned in methods section. The level of significance when p< 0.05.

tumor transplantation. Two of them died during the 4th week, one during the 8th week, one during 12th week and th one during the 13 week of tumor cell transplantation respectively. The animals did not show any apparent toxicity due to formaldehyde. The animals may have died due to other causes. Studies have established decisively that formaldehyde does not have any adverse effect on normal physiology of an organism up to a dosage of 80 mg/kg body weight (Grammer et al, 1990).

Animals following formaldehyde administration did not show any toxic effect The normal animals did not have any toxicity related to formaldehyde at the dose that was used for therapy. The table 1 shows that the hematological profile of animals injected with normal saline and those injected with formaldehyde solution at a dose of 8µg/ml or 118µg/kg body weight (Group C) were in the similar range. Earlier studies have also established that formaldehyde does not have any adverse effects on normal physiology of an organism up to a dosage of 80 mg/kg body weight (7Grammer et al 1990). However, contrarily it was observed, with time, the animals injected with formaldehyde were less susceptible to infection when

compared to controls suggesting a possible improved immune response of the animals bearing the tumor.

DISCUSSION This study reveals for the first time the in vivo anti-cancer property of formaldehyde. Here it is reported that formaldehyde used at a concentration of 8µg/ml or 118µg/kg body weight is very effective in inhibiting tumor cell proliferation and increasing survival without being toxic to animals bearing a poorly immunogenic transplantable tumor. Formaldehyde is a colorless chemical that occurs naturally in the atmosphere through a variety of biological and chemical processes. It is also physically present in the human body at very low concentrations as a result of various metabolic processes. It is water soluble and biodegradable and is known to be associated with minor uneasiness like headache, eye and respiratory irritation, sleep disturbance and increased thirst at exposure levels as low as 0.13 ppm (Thraser et al, 1990]. Chronic exposure to formaldehyde could cause asthmatic symptoms and other reactive airway disease, chronic fatigue, and increased sensitivity to chemicals (Thraser et al, 1990). However, recent studies suggest

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Table 1: Blood profile of animals following formaldehyde administration The table shows the blood profile of animals following formaldehyde administration. The administration of formaldehyde did not show any toxic effect on the animals. The results are shown as Results are expressed as mean ±SEM. The level of significance when p< 0.05.

that there is no adverse effect of formaldehyde on normal physiology of an organism up to a dosage of 80 mg/kg body weight (Grammer et al 1990). Our in vivo studies were based on a formaldehyde concentration of 118µg/kg body weight, which is minimal compared to the suggested dose in literature (Grammer et al, 1990). We have shown that addition of formaldehyde stimulated proliferation of tumor cells in vitro. Recent in vitro studies have also corroborated with the fact that formaldehyde may help in cell proliferation at a minimal concentration of 25µM (Tyhiak et al, 2001; Szende and Tyhiak 2010). In another study, enhanced cell proliferation and reduced apoptotic activity at concentrations of 0.5 and 0.1 mM of formaldehyde was noted (16 Szende and Tyhiak 2010). Our present in vitro study showed that tumor cells behave differently when exposed at diverse range of formaldehyde concentration. At a concentration of 50 ng/ml a significant proliferation of tumor cells was noted compared to control tumor cells, while no proliferation of tumor cells were noted at a higher concentrations of 200 ng/ml and 100 ng/ml, respectively. At this concentration tumor cells were

stimulated to grow at a more rapid rate (17%) compared to non-exposed cells suggesting the possibility of a ‘promoter’ role of formaldehyde on tumor cell proliferation. Thus it is plausible to assume that continuous exposure to formaldehyde for cancer patients may be harmful. This led us to further hypothesize that formaldehyde may act as a ‘promoter’ to already transformed cells which could result in acceleration of tumor burden on continuous exposure. However, at a higher formaldehyde concentration of 200 ng/ml an opposite effect was noted. At this dose formaldehyde was cytotoxic to tumor cells and there was no incorporation of tritiated thymidine at this concentration. Thus the study showed that at a higher concentration it could be suitable for inhibiting tumor growth in vivo. This observation was also supported by the results obtained in the in vtro cytotoxicity assay wherein more than 60% of tumor cell died in presence of formaldehyde by 48 hours of formaldehyde exposure. These cells appeared to be necrotic following exposure to formaldehyde. Similar cellnecrosis in presence of formaldehyde has been noted recently by others (Saito et al 2005; Szende and Tyhiak,

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2010). Tumor cell and endothelial cell culture studies showed that formaldehyde at a concentration of 10 mM caused necrotic cell death (Szende and Tyhiak, 2010) and that formaldehyde and reactive oxygen species (ROS), such as free radicals, are cytotoxic to the cells (Saito et al 2005). Studies with U2OS cells showed that a 50% inhibition of cell growth was noted at 3mM concentration of formaldehyde and this inhibitory effect was influenced by intracellular GSH levels. A higher GSH level protected cells against formaldehyde induced cytotoxicity. It was observed that addition of a GSH precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), had a protective effect on formaldehyde-induced cytotoxicity (Ho et al, 2007]. The free radicals induced GSH reduction, whereas the formaldehyde resulted in the formation of DNA-protein cross-links, with, increase of cellular ROS and cell death was brought about by this combination (Saito et al, 2003). However, It may be interesting to point out that in plants endogenous formaldehyde apparently plays a major role in regulation of normal apoptosis and cell proliferation. These studies including ours clearly establish that formaldehyde has a potential role in regulating cell proliferation and cell death in biological system. Unfortunately, given its apparently adverse chemical nature and lack of knowledge on its biology, no serious studies has been undertaken on its possible use in biological therapy of different pathologies including cancer. Here we have attempted to explore its biological utility and the present data for the first time establishes therapeutic efficacy of formaldehyde in cancer treatment. Our in vivo study clearly demonstrated that formaldehyde at a concentration of 118µg/kg body weight was effective in controlling tumor growth and increasing survival of tumor bearing animals without being toxic to the animals. However, it is to be noted that with increase in the number of tumor cells (x106) in vivo, formaldehyde administration at the tested concentration failed to curb tumor burden. The EAC cells used in this study is not only highly tumorigenic but are poorly immunogenic so further manipulation of the dose could yield better results. Toxicity of formaldehyde becomes a concern for its in vivo application for any therapeutic usage. Earlier studies have shown that administration of formaldehyde at a dose of 80mg/kg body weight did not have adverse effect on body weight, thymus, kidney, liver and cellularity of spleen and lymph nodes. (Grammer et al, 1990). No adverse effect of formaldehyde was noted on normal animals in vivo. Our study clearly demonstrated that formaldehyde did not have any unfavorable effect on the general condition of the animals. On the contrary we observed that formaldehyde seemed to improve the general condition and immune status of the treated animals as the tumor bearing animals were less prone to

infection. It is therefore reasonable to postulate that use of formaldehyde at the suggested concentration may be helpful in improving the health condition of advanced cancer patients which could in turn improve their quality of life that is nevertheless very important for cancer patients. Some recent data have also pointed out the possible therapeutic use of some chemicals like methylated lysine residues and methylated arginine residues in cancer (Szende and Tyhiak, 2010). In our study, formaldehyde treated tumor bearing animals in one group survived for more than 17 weeks after tumor cell transplantation with out showing any signs of toxicity to the chemical. Earlier studies have also established that formaldehyde does not have any toxicity up to a dosage of 80 mg/kg body weight (Grammer et al, 1990]. The toxicity of formaldehyde on cells may be related to its ability to form adducts with DNA and proteins at cytotoxic concentrations which is many folds more than used in our experiments. At higher formaldehyde concentrations, lipid peroxidation also takes place resulting in cell death. (Yoshida et al, 2001). However, there can also be a direct systemic response following formaldehyde administration. Among immune changes reported with formaldehyde exposure, include changes in cell-mediated immunity involving changes in basophiles and/or suppressor cells (NIOSH, 1967). Chronically ill persons after formaldehyde exposure often exhibit immune activation and increased autoantibody formation (NIOSH, 1967; Olsen and Dosing 1982). Formaldehyde also has the ability to stimulate the brain vanilloid receptor that in turn could increase the release of various immune substances (Broughton and Thrasher, 1988). The killing of cells in vivo may be as a result of direct action of formaldehyde or through systemic response. Some studies have shown that cell death resulting from formaldehyde action is necrotic rather than apoptotic (Saito et al 2005). We also have observed that the cells show necrotic features following 48 hours of formaldehyde treatment. Additionally the dead and dying cells could help in generating cytotoxic T Cells (CTL) against the tumor. The apoptotic and necrotic cells are immunogenic and are known to be taken up by dendritic cells (DCs) that stimulate cytotoxic T Cells (CTL) to produce a specific immune response against a tumor (Albert et al, 1998a, Albert et al 1998b). DCs have been shown to efficiently phagocytose dead cells and elicit antigen-specific immune responses (Albert et al 1998a; Albert et al, 1998b, Shaifmuthana et al 2000). It was also demonstrated that Immunization with DCs pulsed with apoptotic tumor cells could prime tumor specific CTLs and confer protection against a tumor challenge in a mouse model (Ronchetti et al, 1999). We also have reported earlier that local irradiation of a tumor followed by cytokine therapy can induce immune response in a metastatic murine lung cancer model (Chakravarty et al, 1997). And using a prostate cancer model, we

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demonstrated that DCs are activated in presence of dead and dying tumor cells (Chakravarty et al, 2007). Thus, the antigens from dead and dying tumor cells can be processed and presented in context of MHC class I by DC that stimulates CD8+ T cells effectively in vitro and in vivo (Sauter and Albert, 2000, Joke et al 2000). Further the dead and dying cells also are effective in immunization protocol (Chakravarty et al, 2006). Formalin-fixed autologous melanoma cells have a potential to function as effective antigen sources for immunotherapy (Obataa et al, 2004]. Similar events may be taking place in the present context wherein formalin induced dead and dying tumor cells may be taken up by DCs, which in turn could generate tumor specific T cells that impacted on survival of animals. We have observed in a different study that formaldehyde treated tumor cells can generate tumor specific immune response (Unpublished data). However, it was noted in a recent study that formaldehyde causes immune suppression only at a higher concentration of 150 ppm in fish (Halladay et al, 2010). The study showed that exposure to formaldehyde at higher concentrations decreased peripheral blood lymphocytes, hematopoietic progenitors and natural killer cell cytotoxicity (Halladay et al 2010). A much lower concentration was used in the effective group in our study. Thus formaldehyde appears to be an enigmatic molecule whose beneficial effect has not been fully exploited. From our study it is established that formaldehyde has a tumor stimulatory role in lower concentration and at an appropriate dose it is therapeutic to animals bearing tumor. Proper manipulation of this molecule could open a new vista in the treatment of cancer in near future.

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