The Anti-Cancer Activity of Rhein in Leukemia - Case Study Example

The anti-Cancer activity of Rhein in Leukemia Introduction Rhei Rhizoma is a traditional Chinese medicine that has been in use for thousands of yearsto treat a range of liver (cholecystitis, cholelithiasis and cholangitis) and pancreatic (acute pancreatitis) disorders (Li and Xiao 1987). As per the Chinese system of medicine, the drug was used to ‘drain fire and free the intestines, cool the blood and resolve toxins, expel stasis and free menstruation’ (Ping et al. 2009). The main active ingredients of Rhei Rhizoma are – rhein, aloe-emodin, emodin and chrysophanol; all of which have shown genotoxic and cytotoxic properties to varying degrees. Rhein, isolated primarily from the roots of rhubarb (Rheum palmatum L. or R. tanguticum Maxim) is an anthraquinone compound (4,5-dihydroxyanthraquinone-2-carboxylic acid) that has caught the imagination of the clinical researchers for its anti-cancer activities. It has been demonstrated to have significant tumorostatic potential in different types of solid tumors, e.g., human glioma (Fanciulli et al 1992), colorectal carcinoma (Gorkom et al. 2002), rat liver carcinoma (Tang et al. 2009), and breast cancer (Lin and Zhen 2009); as well as hematological tumors cell lines, e.g., human promyelocytic leukemia cells (Shigang et al. 2003). Rhein has not only shown a potential to inhibit the tumor cell proliferation by cell-cycle arrest at G0/G1 phase, but it also induces apoptosis in a dose dependent manner. Recently, Chin et al. (2010) have reported that rhein inhibited the cell migration and invasion of human tongue cancer SCC-4 cells and thus could even reduce the incidence of metastatic disease. The anti-cancer activity of Rhein appears to be mediated by a number of mechanisms, some of the prominent ones are – a) Depletion of ATP and inhibition of plasma membrane redox system, thus creating a low energy environment; b) Interference in the cell signaling pathways like MAPK by phosphorylation of the epidermal growth factor receptor, MER and ERK; c) Inhibition of protein kinase C and interleukin-12; d) Blockage of the cell cycle through inhibition of cyclins D3, Cdk4 and Cdk6; e) Increase in the levels of cell cycle/apoptosis regulators like p53, p21 and Bax and reduction in the level of Bcl-2; f) Generation of reactive oxygen species (ROS) that could cause damage to DNA; and g) Inhibition of DNA repair systems like Ataxia Talengectasia Mutated (ATM) gene, BRCA1, DNA-PK and MGMT. Although rhein has shown good anti-cancer potential, it appears to be less potent than the current chemotherapeutic agents, but, since it appears to work through a multiplicity of pathways, it could be a good modulating agent for chemotherapy and might enhance the efficacy of other anti-cancer drugs. The synergetic effect of rhein has been reported - with mitomycin C in hepatoma BEL-7402 and mammary carcinoma MCF-7 cells (Huan and Zhen 2001); with adriamycin on human glioma cells (Gorkom 2002) and with taxol on breast cancer cells xenografts in athymic mice (Lin and Zhen 2009). Inspite of a large number of studies designed to uncover the mechanism of rhein-induced cell cycle arrest and apoptosis, the actual cellular systems involved and the sequence of molecular interactions have not been well defined. Further, studies carried out to see the effect of rhein on hemotological tumor cells are very few and far between. Therefore, the present study is planned to assess the efficacy of rhein on acute lymphocytic leukemia cell lines. Aims and Objectives To demonstrate the anti-cancer efficacy of Rhein. To study the pro-apoptotic effect of rhein on leukaemic cell Lines. To determine the role of cell cycle and cell signaling markers in the mechanism of action of rhein. To compare the therapeutic potency of rhein as an anti-cancer drug with standard chemotherapy agents used in leukaemia. To evaluate the synergistic effects of rhein with the other anti-cancer agents used for treatment of leukemia. Materials and Methods i. Chemicals and reagents Rhein, along with the common laboratory reagents like potassium phosphate, dimethyl sulfoxide (DMSO), Tris-HCl and Triton X-100 would be procured from Sigma Chemical Co. (St. Louis, MO, USA). Cell culture media reagents RPMI-1640, trypsin-EDTA, fetal bovine serum (FBS) and glutamine would be obtained from Gibco BRL (Grand Island, NY, USA). Caspase-3 activity assay kit would be bought from OncoImmunin, Inc (Gaithersburg, MD, USA). ii. Cell culture Leukemia cell lines with specific characteristics viz. acute promyelocytic (HL60), acute lymphoblastic (CEM and MOLT-4) and erythroblastic (HEL-92) would be obtained from the Cancer Research UK Laboratories (London, United Kingdom) and would be maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere with 5% CO2 in air at 37°C. iii. Cytotoxic effect of Rhein with or without other chemotherapeutic agents The viability of the cell lines with or without treatment would be examined by flow cytometry. Rhein at different concentrations (0, 25, 50, 75 and 100 μM) would be used to study its cytotoxic effect. DMSO would be added to the control media, whereas cisplatin C and mitomycin C would be used separately as well as in conjunction with rhein in different culture vials. The cultures would be maintained for 72 hours and observations made by flowcytometry (Becton-Dickinson, San Jose, CA, USA) with an argon ion laser at 488 nm.. To study the effect of rhein and other anti-cancer drugs on cell growth, aliquots would be removed at 6, 24, 48 and 72 hours for assessment of cell number, cell viability, cell cycle distribution and apoptosis. The concentration of rhein required to reduce cell viability by 50% (IC50) would be determined using the sigmoid Emax model (Holford and Sheiner 1981) as follows: Where Ep is predicted effect, Ec is control effect, Emax is maximum effect, C is concentration of drug and n is the sigmoid-fit factor. iv. Determination of Caspase-3 activity PhiPhilux substrate solution would be added to the cell pellet (1×105 cells per sample) and the cells would be incubated at 37˚C for 60 minutes and analyzed with flow cytometry with an argon ion laser at 488 nm. The caspase-3 activity would then be determined as described by Lu et al (2005). To confirm the activation of caspase-3 by rhein to induce apoptosis, the cells would be pretreated with the caspase-3 inhibitor z-DEVDfmk (20 μM) with 50 μM rhein. Apoptosis and caspase-3 activity would then be determined as described above. v. Reactive oxygen species (ROS) estimation The level of ROS of the cell lines would be determined by flow cytometry (Lu et al 2005), using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma). vi. Western blotting for cell-cycle and apoptosis markers In order to study the effect of rhein on Cdk4 and Cdk6, cyclin E and Cdk2, p53, p21, Bax, and Bcl-2 on the leukemia cell lines, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis would be used. Whole cellular protein mixtures would be electrophoresed and exposed to the primary antibodies against the targeted antigens. Horseradish peroxidase labeled anti-species immunoglobulin G1 (IgG1) would be used as secondary antibody and bands would be visualized by electrochemiluminiscence detection system (Amersham Biosciences, UK). vii. DNA analysis for cell-cycle staging Flow cytometry would also be used to observe the cells in different phases of the cell cycle as described by Liu (2005). The percentages of cells in apoptotic fraction, G1, S, and G2/M phases would be determined using the cell cycle analysis program WinMDI v2.4 software. Data Analysis The data would be analyzed using SPSS ver. 15.0 statistical software package. Data would be checked for normal distribution by Shapiro-Wilk testing. If normally distributed, parametric otherwise non-parameteric analyses would be used throughout. Differences between variables and control treatments in culture would be compared by paired Student’s t tests. Ethical Considerations The study has been planned to study the in vitro effect of the Chinese medicine Rhein on Leukemia cell lines. Since there are no experiments planned on animal or human subjects, ethical considerations are limited to doing research in the most honest manner. The results of the research would be beneficial to the humanity as it would add to the knowledge about treating the deadly leukemia. References Fanciulli, M.G., Francesco. P., Bruno, T., Paggi, M.G., Benassi, M.F.A. 1992. Inhibition of membrane redox activity by rhein and adriamycin in human glioma cells. Anti-Cancer Drugs, 3(6), 555-704. Gorkom B.A.P.V., Timmer-Bosscha, H., Jong, Van der Kolk, D.M., Kleibeuker, J.H.  and de Vries, E.G.E., 2002.  Cytotoxicity of rhein, the active metabolite of sennoside laxatives, is reduced by multidrug resistance-associated protein 1. British Journal of Cancer,  86, 1494–1500. Holford, N.H.G. and Sheiner, L.B. 1981. Understanding the dose-effect relationship: clinical applications of pharmacokinetic-pharmacodynamic models. Clin Phamacokin., 6, 429-453. Hsia, T.C., Yang, J.S, Chen, G.W., Chiu, T.H., Lu, H.F., Yang, M.D., Yu, F.S., Liu, K.C., Lai, K.C., Lin, C.C. and Chung, J.G., 2009. The Roles of Endoplasmic Reticulum Stress and Ca2+ on Rhein-induced apoptosis in A-549 Human Lung Cancer cells. Anticancer research, 29, 309-318. Huang, Q., Lu, G., Shen, H.M., Chung, M.C. and Ong, C.N., 2007. Anti-cancer properties of anthraquinones from rhubarb. Med Res Rev., 27(5), 609-30. Li, Y.D. and Xiao, H.X., 1987. Effect of retention enema of da cheng qi tang after gyneco-obstetrical abdominal operation. Zhongxiyi Jiehe Zazhi, 10, 604-605. Liu, W.M., Stimson, L.A. and Joel, S.P., 2002. The in vitro activity of the tyrosine kinase inhibitor STI571 in BCRABL positive chronic myeloid leukemia cells: synergistic interactions with anti-leukaemic agents. Br J Cancer. 86, 472-478. Lu, K.H., Lue, K.H., Liao, H.H., Lin, K.L. and Chung, J.G., 2005. Induction of caspase-3-dependent apoptosis in human leukemia HL-60 cells by paclitaxel. Clin Chim Acta, 357, 65-73. Lin, Y.J. and Zhen, Y.S., 2009. Rhein lysinate suppresses the growth of breast cancer cells and potentiates the inhibitory effect of Taxol in athymic mice. Anti-Cancer Drugs, 20(1), 65-72. Lin, Y.J., Zhen, Y.Z., Shang, B.Y. and Zhen, Y.S., 2009. Rhein lysinate suppresses the growth of tumor cells and increases the anti-tumor activity of Taxol in mice. Am J Chin Med 37(5), 923-931. Ping, R., Feng, Q., Huang.X. and Zhu, Z., 2009. Simultaneous LC Analysis of Aloe-Emodin, Rhein, Emodin, and Chrysophanol in Rhizoma Rhei-Type Preparations. Chromatographia, 70(9-10), 1515-1517. Tang, J.C., Yang, H., Song, X.Y., Song, X.H., Yan, S.L., Shao, J.Q., Zhang, T.L. and Zhang, J.N., 2009. Inhibition of cytochrome P450 enzymes by rhein in rat liver microsomes. Phytother Res., 23(2):159-64. Plan of Work (Gantt Chart) WBS Tasks Start End Duration (Days) % Complete Working Days Days Complete Days Remaining 1 Reagent & Chemicals 22/06/10 28/06/10 7 0% 5 0 5 1.1 Procure chemicals 22/06/10 23/06/10 2 0% 2 0 2 1.2 Reagent Preparation & Calibration of Instruments 23/06/10 25/06/10 3 0% 3 0 3 1.3 Maintain Cell Lines 24/06/10 26/06/10 3 0% 3 0 3 2 Toxicity Experiments 29/06/10 4/07/10 7 0% 5 0 5 2.1 Cytotoxic effects of drugs 29/06/10 1/07/10 3 0% 3 0 3 2.2 DNA Analysis at Cell Cycle stages 2906/10 1/07/10 3 0% 3 0 3 3 Caspase-3 assay 5/07/10 8/07/10 4 0% 4 0 4 3.1 Preincubation with inhibitor 5/07/10 5/07/10 1 0% 1 0 1 3.2 Flowcytometry 5/07/10 7/07/10 3 0% 3 0 3 3.3 Caspase-3 Assay 8/07/10 8/07/10 1 0% 1 0 1 4 Western Blotting 12/07/10 15/07/10 4 0% 4 0 4 4.1 Protein extraction 12/07/10 12/07/10 1 0% 1 0 1 4.2 Gel Preparation 13/07/10 13/07/10 1 0% 1 0 1 4.3 Electrophoresis and Blotting 14/07/10 15/07/10 1 0% 1 0 1 4.4 Electrophoresis and Blotting 14/07/10 15/07/10 1 0% 1 0 1 5 Data Analysis & Writing 19/07/10 23/07/10 4 0% 4 0 4 COSHH Risk Assessment Substances used which have potential to cause harm Chemical Hazard Identified OEL (If assigned) Precautions in Handling Emergency Action Acrylamide/Bisacrylamide Toxic, harmful, irritant, carinogen, teratogen OEL 0.3 mg m2 Double layer of nitrile gloves Use in fume hood Do not use if pregnant Storage – keep lid tightly closed, refrigerate, keep in dark Spillage – evacuate area, absorb with paper toweil and place in closed container. Dispose in yellow bin. Ventillate area and wash spill site after pickup is complete. * First aid- see below Dimethyl Sulfoxide (DMSO) Inflamable, can cause nausea, skin rash and specific (garlic-onion-oyster) body odor - Keep away from sources of ignition. No Smoking. Do not breathe vapor or mist. Avoid contact with skin, eyes, or clothing. Butyl rubber or nitrile (NBR) rubber gloves. Eye goggles If inhaled, move to fresh air, treat symptomatically * First Aid: Skin contact – flush with copious amounts of water for at least 15 minutes. Remove contaminated clothing and wash before reuse. Unless contact has been slight obtain medical attention. Inhalation – remove to fresh air, rest and keep warm. If breathing is difficult give artificial respiration and obtain medical attention. Ingestion – do not induce vomiting. Provide water for person to thoroughly wash out mouth (and sip if required). Obtain medical attention. Read More