Epirubicin (PHARMORUBICIN®), anthracycline antibiotic, inhibits topoisomerase II which is essential for DNA replication, chromosome condensation and chromosome segregation. Inhibitors of topoisomerase II are important drugs used in the therapy of many neoplasms including breast cancer, lung cancer, testicular cancer, lymphomas and sarcomas. Similarly, cyclophosphamide (CYTOXAN®) is also a cytotoxic drug by cross-linking of strands of DNA and RNA with their alkyl group. So, it is known as an alkylating agent. It is also used in breast cancer, leukemia, lymphoma, Ewing’s sarcoma, lung cancer etc. But, since cyclophosphamide is a prodrug which requires hepatic enzymes activation to form active metabolites, it does not work their cytotoxic action in vitro. In our paper, we identified the cytotoxic actions of both drugs in vitro by using the chemosensitivity assay. In this assay, we used MCF-7 breast cancer cell line. As a result, we found that the amounts of epirubicin are significance correlated with the percentage of cancer cell survivals. On the other hand, cyclophosphamide does not show proper effect on the breast cancer cell lines. In daily practice, these two drugs are widely used in many cancer therapies. Thus, we conclude that cyclophosphamide needs in vivo activation to express their oncolytic action otherwise epirubin does not need.
Introduction Response of the various tumour cell lines (i.e. breast, colon, lung, prostate etc.) to the chemotherapeutic agents are evaluated by chemosensitivity assays. A chemosensitivity assay is a vitro laboratory test that can identify new compound or drug with anti-tumour properties. This type of study demonstrates that tumour resistance was predicted with greater accuracy than sensitivity. Chemosensitivity assay may help in choosing the best drug or compound for the cancer being treated. Nowadays, there are many different types of chemo sensitivity assays to access the sensitivity have been developed. However, procedure of this assays are basically more or less the same. First of all, cancer cell line is extracted from the culture media and incubation of these cancer cells with tested drugs or compounds. Then, assessment of the cell survival and interpretation of the observation have been done. Therefore, generally for the chemo sensitivity to be done, the specific cell line to be assessed and cell cultures are required. There are a lot of cell lines. Firstly, primary cells which are explanted directly from a cancer patient cannot grow continuously in vitro and die eventually whereas the Secondary cells also from a donor. But their physical characteristic may change after they die. Another one is immortalized cells also known as transformed cells which can grow and divide in vitro when the appropriate conditions are maintained. Besides, eukaryotic cell lines are more difficult to culture compared to the prokaryotic cells.
In our study, the aim is to identify compounds (Epirubicin and cyclophosphamide) which have activity against a specific type of cancer (breast) have a novel mechanism of action. We use breast cancer cell line MCF-7 (Michigan cancer foundation-7). MCF-7 cells are oestrogen receptor positive control cell line and basic cell line of breast cancer. Moreover, we use MTT assay which is colorimetric assay, sensitive in vitro for measuring cell proliferation or apoptosis. MTT is added into the culture plate and incubated. This compound is reduced by mitochondrial reductase which is present in active living cells to purple colour formazan crystals. Therefore, this reduction reaction is taken place only when reductase enzymes are active.
Acidified ethanol solution dissolves these crystals into colored solution. This solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. So, the reduction rate of tetrazolium is directly proportional to the rate of cell proliferation.
Epirubicin is an anthracycline antibiotic which is commonly used anti-neoplastic agent. Epirubicin is produced from Streptomyces Peucetius. It inhibits topoisomerase II which is essential for DNA replication, chromosome condensation and segregation. Epirubicin intercalate into DNA and forms a complex with DNA resulting in inhibition of DNA and RNA synthesis. It also generates reactive metabolites. So, these cytotoxic free radicals interact with many intracellular molecule and cell membranes. Thus, the biologic effects of the epirubicin may not be based solely on topoisomerase II activity.
Structure of Epirubicin
Epirubicin have some adverse effects. Among them, dose related myelosupression is the most common and even fatal. Also, it has cardio-toxic effect. Thus, it is contraindicated in severe cardiovascular diseases. But, side effects of epirubicin are relatively lesser than other anthracyclines. Besides, it has potential mutagenic and teratogenic effects.
Cyclophosphamide (prodrug) is an alkylating agent of the nitrogen mustard type. It is an inactive cyclic phosphamide ester of mechlorethamine. Inactive form transforms to an alkylating metabolites (Aldophosphamide). A small proportion of aldophosphamide is converted into phosphoramide mustard (active form) and acrolein by hepatic microsomal oxidation system (Cytochrome P450 enzymes) and some peripheral activation. Phosphoramide mustard alkylates or binds to DNA/RNA and then cross-linking of strands of DNA and RNA. Its action do not appear to be cell-cycle specific. Cyclophosphamide can prevent cell division.
It has some adverse effects especially haemorrhagic cystitis (up to 40%) which may be due to acrolein direct injury to the urothelium. Other significance side effects are myelosupression and secondary malignancies. Thus, cyclophosphamide is contraindicated in severe leucopoenia, thrombocytopenia, hepatic or renal dysfunction. It also has potential risk of congenital malformations when using in pregnancy.
Materials and Method Materials MCF – 7 (Michigan cancer foundation -7)
96 Well culture microtitre plates
RPMI 1640 medium (Roswell Park Memorial Institute)
MTT assay proliferation kit
DMSO (Dimethyl Sulfoxide)
Universal Microplate Reader (Spectrophotometer)
Filtered Fume Enclosure
Method MCF-7 breast cancer cell line is provided. First of all, the cap of MCF-7 cultured flask is opened and discarded the medium. 5ml of PBS (phosphate buffered saline) is added to wash this flask and allowed to mix then removed the PBS. 1-2ml of trypsin is added and keep the flasks on 1-2 minutes permitting to detach the cells. If required, tap periodically the flask from the bottom. After that, the whole solution is transferred to the 15ml falcon tube and then, centrifuged with 1200 rpm for 1-2 minutes. The supernatant is removed leaving behind the cell pellet. 180Î¼l of cancer cells (MCF-7) are put into the 96 wells microtitre plates from lane 2 to 12. After this, the microtitre plates are incubated at 37’C with 5% CO2, 95% air and humidity for 24 hours before any addition of the experimental drugs. 200Î¼l of RPMI 1640 containing 5% foetal bovine serum and 2mM L-glutamine is also added into the lane 1 and 20Î¼l to each well of lane 2 creating negative control and positive control respectively. 1µM of 20 µl of epirubicin is added to the lane 3 (row A to H) and 20µl (0.5 µM) of epirubicin is also added to the lane 4. Similarly, from lane 5 to 12, decreasing concentration of the drug with same amount is placed (0.25µM, 0.125µM, 0.0625µM, 0.0312Î¼M, 0.0156Î¼M, 0.0078Î¼M, 0.0039Î¼M and 0.0019Î¼M respectively). After finishing, the plate is labelled and incubated at 37’C for 4 days. Repeating the same procedure is done for cyclophosphamide. Overall procedure should be done under filtered Fume Enclosure to prevent contamination. Moreover, these two microtitre plates are stored wrapping with aluminium paper to avoid light because both drugs are photosensitive. After 4 days later, 20 µl of MTT (5mg/ml) is added to each well of the microtitre plates and cultured for 4 hours at 37’C for cleaving MTT. After 4 hours, all the solution (medium or drugs and MTT) within the plates is discarded carefully. Then, each well is filled with 150µl of DMSO to dissolve the crystals of formazan and mix the solution properly with stir bars. Finally, optical density (OD) of each well is read by spectrophotometer (Universal microplate reader) at 490nm wavelength.
Discussion The respective tables and graphs show how to response the two drugs, Epirubicin and cyclophosphamide, on the MCF-7 breast cancer cell line. In graph 1, epirubicin killed the significant amount of the cancer cells at the concentration of 1 µM. In this concentration, only 12.58% of the cancer cells are survived. Then, percentage of cell survival rates are gradually increased from lane 4 onwards while drug concentrations are also two fold diluted. Therefore, percentage of cell survival is inversely proportional to the epirubicin concentration. There is obviously seen that epirubicin has direct cytotxic effect in vitro. From graph 1, we assumed the IC50 (half maximum inhibitory concentration) which is about 0.125Î¼M. IC50 is a measurement of effectiveness of compound or drug in inhibiting a biological or biochemical function. This quantitative measure indicates amount of a particular drug or other substance is needed to inhibit a given biological process by half. Epirubicin is one of the compounds of anthracycline antibiotic and it can inhibit DNA topoisomerase II which is required for DNA break in replication. So, it effect on ‘S’ phase of cell cycle. Moreover, anthracyclines can undergo one and two-electron reduction, since they are members of the quinone family, producing reactive compounds that damage macromolecules and lipid membranes. Epirubicin is mainly metabolized in the liver and excreted predominantly in the bile and then faeces. It can cross placenta and can be found in breast milk. Conclusively, we can assume that the cytotoxicity effect of epirubicin is still active in vitro chemosensitivity assay.
Next, according to the table 2, percentage of cell survival is not relevant with the concentration of the drug, cyclophosphamide. Apart from minor differences, there is no significant change in cell survivals. Similarly, in the graph of cyclophosphamide, the trend of cancer cell survivals is also swinging across the drug concentration but not correlated with cyclophosphamide concentration. Cyclophosphamide is the alkylating agent but it is prodrug. It is inactive in vitro. So, it needs to be activated with the action of hepatic and intracellular enzymes to form active one (4-hydroxycyclophosphamide, aldophosphamide, acrolein and phosphoramide mustard). Only active form can prevent the cell division by crossing-linking DNA (especially guanine base) and RNA strands with their alkyl group. So, it disrupts the DNA replication and subsequent steps. Cyclophosphomide is cell cycle non-specific drug. It is metabolized by liver enzymes (cytochrome P450) and excreted in bile. Therefore, main cytotoxic action of cytophosphamide is solely depended on the action of cytochrome P450 (CYP 450). Because of this reason, we can assume that cyclophosphamide has little or no cytotoxicity in vitro.
Chemo-sensitivity assays are intended to predict the sensitivity of in vitro cancer cell line to chemotherapeutic agents and identify more effective treatment protocols. So, the chemo-sensitivity assay may help in choosing the best drug or drugs for the cancer being treated. Despite there are many ways to analyse the effects of drugs on cell metabolism and cell morphology, chemosensitive assays can be utilized routinely in the clinical experiments. In our assay, MTT is used. It can measure cell proliferation, apoptosis and cell survivals. MTT compound is added into the MCF-7 cells cultured plates which is treated with anti cancer drugs (epirubicin or cyclophosphamide). These MTT is reduced by metabolically active cancer cells to insoluble purple formazan crystals. The rate of reduction reaction is proportional to cell survivals. So, we can calculate the optical density of formazan with spectrophotometer. Besides, for effective assays, proper handling and certain precautions are strictly essential. Otherwise, there will be errors in the assays and difficult to read the absorbance reading and the data interpretation will be inappropriate. Furthermore, proper experiment has to be done under aseptic condition as much as possible. Microtiter plates containing drugs and cell line need to store in the dark because most of the anti cancer drugs are photosensitive. Similarly, MTT should also be store at dark. It is also important to check the colour of the reagent before we use. However, there might be some problems and errors that likely to occur in the assays. If the MTT is in blue or green colour, it is likely to be contamination with the cellular or bacterial culture. If the absorbance readings are too low, this might probably, the cells are not proliferations because of improper culture conditions, inadequate cell recovery time after plating or errors in the procedure.
In these days, there are many methods for anti cancer drug screening. The national cancer institute (NCI) use the COMPARE method in vitro. In this method, a probe or seed compound can be specified by using the NCI compound’s accession number (NSC number). And then compare algorithm is carried out to evaluate the entire database in the order of the similarity of the responses of the cell lines to the seed compound. By applying this algorithm, it is possible to assign mechanism of action to test or determine a compound or to determine that the response pattern is unique and not similar to that of any of the standard prototype compounds included in the NCI database.
DTP anti-cancer Screening Paradigm Test request
(Drugability, novelty, reproducibility, potency, selectivity, mechanism)
Acute Toxicity (Max. Tolerated Dose)
Hollow Fiber Assay
Adopted from http://dtp.nci.nih.gov/docs/misc/commonfiles/ACSPFlowChart.pdf
The screening is a two-stage process, starting with the evaluation of all compounds (single dose of 10Î¼M) against 60 different human tumour cell lines representing leukaemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. The outcome is then reported to analyze by the COMPARE program. Moreover, in vitro identification of potential anticancer agent is required to enhance with demonstration of in vivo animal models for screening the preclinical development. Then, the hollow fibre assay can be used for in vivo animal models. It is a current initial in vivo screening activity for the potential anticancer drugs with cytotoxicity in vitro screening. On the other hand, when potential anticancer drug screened in vitro needs to demonstrate for in vivo efficacy, xenograft models are used to test the efficacy. NCI currently research with developmental Therapeutics Program (DTP) Human Tumour Xenograft Models. The compounds are examined for anti tumour activity in human tumour xenograft model in nude mice or rodent tumour models after screening with NCI 60 cell lines and hollow fibre assay.
Conclusion In conclusion, the anticancer drugs, epirubicin and cyclophosphamide sensitivity are assessed in vitro with MCF-7 breast cancer cell line by using MTT chemo sensitivity assay. Cytotoxic effect of these two drugs is different in vitro assay, although these two drugs play an important role in anticancer regime. As a result, epirubicin has significant cytotoxic effect on MCF-7 cell line in vitro whereas cyclophosphamide does not. Cyclophosphamide is prodrug which needs to be activated by cytochrome P450 (hepatic enzymes) in vivo. So, the whole study suggests that their cytotoxic effects differ significantly in vitro. Anyway, the chemo-sensitivity assay is important and useful in vitro method for research to develop anti-cancer drugs, their efficacy and therapeutic effects.
Role of Bacteria in Cancer Treatment
Cancer can be promoted by several pathogenic bacteria to initiate abnormal cell growth by attacking the immune system or suppressing apoptosis (Mager 2006). However, bacterial toxins can still be used for tumor suppressor and cancer vaccines on immunotoxins of bacterial origin (Patyar et al. 2010). Bacterial colonization of tumors was initially attributed to the hypoxic nature of solid tumors (low O2 levels). It has been proposed that the anaerobic nature of hypoxic/necrotic regions within tumors promotes growth of anaerobic and facultatively anaerobic bacteria. Areas of necrosis may also provide nutrients such as purines to further promote the growth of bacteria. The use of genetically modified and virulence reduced bacteria for destruction of tumors, and bacterial gene-directed enzyme prodrug therapy have shown promising potential in the development of bacteria therapy. (Patyar et al. 2010) Subsequently, it has been realized that anaerobic bacteria can selectively grow in tumors. However, these bacteria were not suitable for cancer therapy because of their high pathogenicity. Later on studies in animal models revealed that obligate anaerobic bacteria such as clostridia species proliferate preferentially in necrotic and therefore anaerobic regions of solid tumors. This actually resulted in tumor regression but was accompanied by acute toxicity and most animals became ill or died.
A wide range of gene therapy strategies exists aimed at inducing malignant cell death, be it directly or indirectly.
Direct cell killing. The most direct gene therapy strategy to treat tumors involves introducing a vector and gene to a malignant cell that directly induces death of that cell. There are a number of mechanisms by which this can be achieved, including the delivery of genes cytotoxic to the cell (pro-apoptotic genes or so-called ‘suicide genes’) or through oncolysis induced by the bacterial vector itself (as is observed with Clostridium and Salmonella).
Oncolytic vectors. The oncolytic approach uses replication competent bacteria that are capable of spreading through the tumor tissue to infect neighbouring cells, with cancer cells killed as a result of infection. Therapeutic trials employing clostridial species mainly rely on the natural oncolytic activity of the vector to achieve tumor therapeutic responses. Following IV administration, clostridial spores germinate within tumors, killing cancer cells as they replicate, and have been shown to produce significant oncolytic effects in preclinical and clinical studies.
Efforts to refine the process have involved pre-treatment measures to make the tumor environment more hypoxic, combination therapies and more recently, genetic engineering. However, clostridia are typically difficult to manipulate genetically, which has hampered their development in terms of expression or delivery of heterologous genes. Only recently have strains been engineered to encode additional heterologous genes, aimed at enhancing the therapeutic effect (see below). Nonetheless, there is still cause for optimism with this treatment strategy in line with further improvements in genetic technologies for the species.
Cytotoxic genes. Bacterial vectors can mediate expression of agents that are cytotoxic to the host cell. The extracellulardomain of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent apoptotic agent in tumor cells, with minimal toxicity to normal cells. Attenuated S. typhimurium has been used to express TRAIL under the control of the prokaryotic radiation-inducible RecA promoter, with systemic administration of this vector resulting in xenograft tumor reduction. B. longum has also been utilized to express this agent within murine tumors resulting in significant regression. Indirectly cytotoxic genes have also shown promise.
Prodrug activating genes (suicide genes) encode a protein that is capable of directly or indirectly causing cell death. While some suicide genes express products that are directly toxic for the cell, e.g., Diphtheria toxin or Pseudomonas exotoxin, the best known agents encode enzymesthat convert non-toxic pro-drugs into highly toxic metabolites.
Gene-directed enzyme prodrug therapy (GDEPT) is a twostep approach. In the first step, the transgene is delivered into the tumor, while in the second step, a prodrug is administered which is selectively activated by the expressed enzyme. The most widely used system is the thymidine kinase gene of the Herpes Simplex Virus (HSVtk) in combination with the prodrug ganciclovir.
HSVtk phosphorylates ganciclovir to produce a cytotoxic metabolite. Other systems include the cytosine deaminase (CD) gene in conjunction with 5-fluorocytosine. A number of bacterial vectors have been successfully utilized to deliver suicide genes as summarized in Anti-angiogenic therapy. Angiogenesis is the formation of new capillary blood vessels from existing microvessels. For cancer therapy, strategies based on the manipulation of angiogenesis are referred to as anti-angiogenic strategies and seek to prevent new vessel formation or to inactivate pre-existing vessels. Gene-based anti-angiogenic therapy holds the potential to provide long-term anti-angiogenic protein production, and can be readily used in conjunction with other strategies. Endostatin is an endogenous inhibitor of angiogenesis, first discovered in 1997. It suppresses endothelial cell proliferation and acts as a competitor of angiogenic inducers secreted by tumor cells, such as fibroblast growth factor and vascular endothelial growth factor. However, results with administration of recombinant endostatin protein in clinical trials have been disappointing, due to poor solubility of the protein, in addition to the requirement for long-term multiple administrations.
Upregulating the immune system. Cancer immunotherapy approaches concentrate on killing the tumor cells through direct or indirect intervention of various effector cells of the immune system, which include antibody-producing B cells, CD8 CTL, CD4 helper T cells, and NK cells. Gene therapy can be employed to induce tumor or other cells to produce immune upregulating cytokines that can attract and enhance anti-tumor activity of various lymphocytes. S. typhimurium has been used in several murine trials examining immunotherapies, with significant tumor reduction resulting from local bacterial expression or tumor cell expression of the immune-stimulating molecules IL-18, CCL21, LIGHT or Fas ligand.96-99 Preclinical studies have also used bifidobacteria in combination therapy with cytokines such as granulocyte colony-stimulating factor (GCSF), resulting in superior anti-tumor effects. Interestingly, the immune response was primarily directed against tumor cells rather than the bacterial vector cells.
DNA vaccination. The goal of cancer vaccines is to break tolerance of the immune system to specific antigens known to be expressed mainly or exclusively by particular tumor cells. DNA vaccines expressing a defined tumor antigen have shown significant promise both preclinically and in clinical trials. This strategy involves delivery of a vector that expresses the gene of interest, and functions to target immune activity in a similar manner to which traditional vaccines work. Vaccination strategies attempt to stimulate immune responses by generatingcytotoxic T lymphocytes and/or antibodies from B cells to break the pre-existing tolerance to specific antigens. Bacteria that target inductive cells of the immune system are highly attractive candidates for vaccine delivery and have been developed as live vehicles for inducing protective responses to a wide variety of antigens. Members of the Salmonella genus have been widely used as antigen carriers and several well-characterized safety attenuated strains are available.105-108 Salmonella is capable of triggering both humoural and antigen-specific T-helper and cytotoxic responses. S. typhimurium vectors deliver transgenes to the body via the monocyte cell population. After oral intake, the bacterial vector cells are phagocytosed by monocytes in the intestine. The monocytes differentiate and migrate to lymph nodes and the spleen. Finally, the attenuated auxotrophic S. typhimurium (unable to replicate in mammals) lyse and release plasmid into the cytoplasm of monocytes, followed by expression of the desired antigen and presentation to the immune system. This delivery platform has shown success in several preclinical tumor models employing various tumor antigens. A number of live attenuated strains of Listeria have been developed expressing a broad range of tumor antigens, such as Her-2/neu (an oncoprotein associated with a wide variety of cancers, Melanoma Associated Antigen (MAGE)117 and prostate specific antigen (PSA).118,119 The cytoplasmic location of L. monocytogenes is significant as this potentiates entry of the antigen into the Class I MHC antigen-processing pathway leading to priming of specific CD8 T-cell responses. IV administered attenuated L.monocytogenes expressing HPV16 E7 was recently used in phase I clinical trial on patients with metastatic cervical cancer.120 Apart from some flu-like symptoms and fever-related hypertension in some patients, the vector was well tolerated. In addition, 30% tumor reduction was noted with an increase in overall survival, indicating the safety and efficacy of listerial vectors in patients and paving the way for clinical development of this vector strategy.
Much of the current research intended to achieve selective replication within, and lysis of, tumor cells has focused on viruses, but recent observations in murine models with facultative anaerobic bacteria (1), as well as data generated more than 30 years ago with obligate anaerobic bacteria (2), indicate that some bacterial species can also preferentially replicate and accumulate within tumors. In contrast to viruses, the bacteria reside primarily in the extracellular tumor microenvironment (3) and possess certain features that may be advantageous in the treatment of cancer. Thus, bacteria are motile, which facilitates their spread throughout the tumor and can help target systemic disease. Because of their large genome size, bacteria can readily express multiple therapeutic transgenes, such as cytokines or pro–drug-converting enzymes, and their spread can be controlled with antibiotics if necessary.
Bacteria can be used as tumoricidal agents which can suppress or destruct the tumor via direct tumoricidal effects or by delivering the tumoricidal molecules to the tumor site(Patyar et al. 2010). Experimental studies have shown that pathogenic species of the anaerobic clostridia were able to proliferate preferentially within the necrotic (anaerobic) regions of tumors in animals as compared to normal tissues thus resulting in tumour regression but was accompanied by acute toxicity and most animals became ill or died Deletion of two of its genes –msbBandpurI-resulted in its complete attenuation (by preventing toxic shock in animal hosts) and dependence on external sources of purine for survival. Bacteria can also act as vector for gene therapy. Bacteria as carriers of tumoricidal agents.
Bacteria is genetically engineered to express some therapeutic gene(Patyar et al. 2010). Bacterial toxins can cause cell death by alter cellular processes that control proliferation, apoptosis and differentiation(Patyar et al. 2010). Bacterial toxins have to some extent already been tested for cancer treatment. Bacterial toxins can kill cells or at reduced levels alter cellular processes that control proliferation, apoptosis and differentiation. These alterations are associated with carcinogenesis and may either stimulate cellular aberrations or inhibit normal cell controls. Cell-cycle inhibitors, such as cytolethal distending toxins (CDTs) and the cycle inhibiting factor (Cif), block mitosis and are thought to compromise the immune system by inhibiting clonal expansion of lymphocytes. In contrast, cell-cycle stimulators such as the cytotoxic necrotizing factor (CNF) promote cellular proliferation and interfere with cell differentiation. Bacterial toxins binding to tumor surface antigens
Bacterial toxins conjugated to ligands. This process is accomplished by eliminating binding to toxin receptors by conjugating the toxins to cell-binding proteins such as monoclonal antibodies or growth factors. Bacteria also act as immunotherapeutic agents as bacteria can promote the antigenicity of tumor cells and prevent the tumors to escape the immune system even they are weekly immunogenic(Patyar et al. 2010). Thus one of the novel immunotherapeutic strategies employs bacteria to enhance the antigenicity of tumor cells. The majority of all the anaerobic bacteria discussed so far can form highly resistant spores which allow them to survive even in oxygen-rich conditions, although they cannot grow or multiply there. But once they meet favourable conditions, such as the dead areas inside tumors, the spores can germinate and the bacteria thrive, making them ideal to target cancers.