Assignment Sample on The Effects Of Combinational Therapy
Introduction
A certain condition when the number of white blood cells is increased in the human body is referred to as leukemia. Leukemia is a group of blood cancers characterized by the abnormal production of white blood cells in the bone marrow. While traditional chemotherapy, viz doxorubicin, is effective in killing cancer cells, it often leads to severe side effects and drug resistance. This has led researchers to explore combinational therapies to improve treatment outcomes and reduce toxicity. MDIVI-1 is a novel combination that has shown neuroprotective properties by targeting mitochondria-associated events. This study depicted doxorubicin, which is an anthracycline chemotherapy drug that works by intercalating DNA, inhibiting DNA topoisomerase II, and generating free radicals, ultimately leading to cell death.
1.2 Background of the research
Combinational therapy has appeared as an advantageous approach to treating different types of cancers, including leukemia. One such combination that has shown possibility is the usage of MDIVI-1 with doxorubicin in leukemia treatment. Recent studies have suggested that it could have potential applications in cancer therapy as well. When combined with doxorubicin, MDIVI-1 has demonstrated synergistic effects in leukemia treatment. The proposed mechanism of action involves the unique properties of both MDIVI-1 and doxorubicin.
Figure 1: Attenuation of Doxorubicin-Induced Cardiotoxicity by mdivi-1
(Source: https://journals.plos.org)
MDIVI-1 marks mitochondrial function, which recreates a critical role in cell survival and apoptosis (programmed cell death). By hampering the procedure of mitochondrial actions, MDIVI-1 sensitizes leukemia cells to subsequent treatment with doxorubicin. Yet, cancer cells can formulate resistance to doxorubicin over time. When MDIVI-1 is distributed alongside doxorubicin, it improves the drug’s cytotoxic effects on leukemia cells (Esmaeili et al. 2021). This mixture not only enriches cancer cell death but also facilitates the dosage of doxorubicin needed, thereby decreasing its adverse effects on normal tissues. Similarly, leukemia stem cells, which are in charge of disease relapse and progression, may be targeted by the combined therapy of doxorubicin and MDIVI-1. Combination therapy may produce a longer-lasting and more sustainable remission by targeting these stem cells. Preclinical studies’ first findings are encouraging, but more investigation is required to fine-adjust the dose, evaluate potential adverse effects, and confirm the safety and effectiveness of this combination in human clinical trials. If effective, MDIVI-1 and doxorubicin combination therapy might completely alter the way leukemia is treated and open the door to cutting-edge methods for treating other cancers.
1.3 Research Aim and Objective
This section mainly provides various types of aims and objectives based on the particular research topic. The goal of this study is to look at the efficacy of combining MDIVI-1 alongside doxorubicin in the treatment of leukemia (Liang et al. 2020). The four objectives based on the research are mainly given below.
- To establish each of the cytotoxic impacts of MDIVI-1 as well as doxorubicin upon leukemia cells.
- To assess mixed effects regarding MDIVI-1 as well as doxorubicin upon leukemia tissue viability
- To examine the impact of MDIVI-1 upon doxorubicin-induced cardiotoxicity.
- To investigate possible mechanisms that underlie the effects that have been observed.
1.4 Research Question
This section mainly describes the questions of the research based on the effects of combinational therapy of MDIVI-1 with doxorubicin in the leukemia research topic. So the research question is given below (Genovese et al. 2021).
- What constitutes the cytotoxic impacts of MDIVI-1 as well as doxorubicin upon leukemia cells individually?
- Does the combination of MDIVI-1 as well as doxorubicin have a synergistic impact on leukemia cell viability?
- How can MDIVI-1 affect doxorubicin-induced cardiotoxicity during the course of therapy of leukemia?
- What might be the biological processes behind the reported effects of combination treatment on leukemia cells?
Research Significance
The study of MDIVI-1 combination treatment with doxorubicin within leukemia is relevant in the realm of cancer-related therapies. Leukemia is a difficult disease with few treatment choices, and drug resistance frequently affects the efficacy of current medicines. This unique combinational method under research has an opportunity to overcome medication resistance and improve treatment results (Yun et al. 2020). If the combination of drugs proves to be highly synergistic, it might lead to enhanced leukemia cell elimination while potentially lowering individual medication dosages, and decreasing side effects. This has the potential to greatly improve the quality of existence for leukemia patients receiving therapy. Comprehension of the underlying processes along with shifts in resistance to drugs patterns may give vital insights into the way these medicines interact and may aid in the creation of customized treatment methods for various leukemia subtypes. The discovery has the possibility to enhance leukemia management, providing hope for more successful and tailored medicines in the battle against this dreadful illness.
1.6 Research Rationale
The rationale for researching MDIVI-1-doxorubicin combination therapy in leukemia arises from the critical requirement to improve therapeutic efficacy as well as overcome drug resistance. Due to its complexity with the restrictions of current therapeutic techniques, leukemia poses substantial obstacles. Drug resistance continues to be a key barrier, resulting in failure of therapy and disease return (Anderson, 2021). Leukaemia cells rely largely upon mitochondrial function for viability as well as proliferation, MDIVI-1’s specific strategy of blocking mitochondrial division may provide an advantage. MDIVI-1 can selectively trigger apoptosis within cancer cells despite sparing cells that are healthy via altering mitochondrial dynamics. The combination of MDIVI-1 and doxorubicin, an extensively utilized chemotherapeutic drug, is especially appealing due to their complementary modes of action [Referred to Appendix 1].
Doxorubicin targets nuclear DNA predominantly, and when combined with MDIVI-1, which influences mitochondrial motion, both drugs have the ability to strike both nuclear as well as mitochondrial elements in leukemia cells. This synergistic method may result in improved anti-cancer properties as well as decreased medication resistance, potentially boosting therapy efficacy and improving patient outcomes. In addition, research into this mixed therapy in leukemia is important since it might provide a fresh and customized treatment method for leukemia patients (dos Santos Arruda et al. 2019). Identifying the connections between these substances can provide knowledge about their ways of action as well as drug resistance trends, leading to the development of personalized treatment regimens to optimize therapeutic advantages while minimizing side effects in the therapy of leukemia. This study has the possibility to make a significant contribution to advances in leukemia treatment as well as care for patients.
1.7 Summary
Here mainly conclude that leukemia is an important global health issue, including medication resistance as well as adverse effects complicating therapy. Combinational treatment has come to prominence as a possible solution to these challenges. This study investigates the effectiveness of MDIVI-1, the mitochondrial division inhibitory substance, in conjunction with doxorubicin, a commonly used chemotherapy medication, in the treatment of leukemia. The study’s goal is to look into the synergistic advantages of the two therapies in terms of treatment performance, medication opposition; as well as adverse reactions. This research holds potential for improving leukemia management offering hope for more successful and personalized treatments through investigating the cytotoxic impacts of both medications on leukemia tissue cells, analyzing the effects on the viability of cells, as well as discovering probable pathways.
2.0 Materials and Methodology
2.1 Materials
MTT assay-
- Cells isolated from the rabbits
- MTT assay cell plate
- Tetrazolium bromide, 98%
- Phosphate buffer, pH 6.5 (Song et al. 2022).
Western Blotting-
- PIERCE fast western blotting kit
- SUPER SIGNAL west femto, Rabbit; for thermo scientific part.
- Fast western antibody diluent, 200 ml
- Rabbit optimizes HRP reagent.
Flow cytometry assay-
- Annexiv vs. apoptosis detection kit- BD Pharmingen
- 10x annexed vs binding buffer
- Propidium iodide staining solution
- FITC annexin V [compound number 51]
Figure 2: Metabolism-Based Therapeutic Strategies Targeting Cancer Stem Cells
(Source: https://www.frontiersin.org)
2.1.1 MTT assay
- At first, cells isolated will be taken, and the required cell concentration should be achieved by incubating the cell culture for 24 to 48 hours at 370 C (TarvijEslami et al. 2023).
- Then, different concentrations of cell cultures were prepared. Dilutions up to 10-6 is prepared.
- After that, 1 ml of culture was taken and 100 μl of the sample was poured into the wells of the plate.
- Next, 1 μg/ml actinomycin C1, and different amounts of hTNF-alpha in the concentration of 0.5 mg/ml were added to each microplate well-containing cell culture.
- The cell culture was incubated at 370 C for 24 hours.
- Then, 10 μl of MTT labeling reagent was added to each well, and the plate was incubated for 4 hours in humid conditions.
- Next, 100 μl of solubilization solution was added into each well, and incubated overnight at 370 C (García-Heredia and Carnero, 2020).
- At last, the OD (optical density) or absorbance of the sample was taken in an ELISA reader.
The MTT test is a common technique for determining cell viability as well as proliferation. Cells obtained from rabbits are going to be utilized in this investigation. To obtain the appropriate cell concentration, the cells will be grown and incubated. For therapy, various concentrations of cultured cells will be created. MDIVI-1, doxorubicin, along with additional drugs will be administered to the cells in various combinations. Following incubation, an MTT labeling solution will be applied to each well and absorbance will be determined with an ELISA reader. This experiment will aid in determining the combination’s synergistic effects on leukemia cell survival.
2.1.2 Flow cytometry assay
- Cell suspension was collected, and placed into the falcon tubes. Then, centrifuged at 1000 rpm for 5 minutes.
- The cell pellet was resuspended into 1-2 ml of PBS buffer solution.
- After discarding the supernatant, the cell culture was incubated for 20 mins. At 370
- Then, the cell sample was again centrifuged at 1000 rpm for 5 minutes.
- The cell pellet was resuspended in the FLICA solution and incubated at 370 C for 1 hour. The culture was gently agitated every 20 minutes (Alexa-Stratulat et al. 2019).
- Then, 2 ml of PBS buffer was added and again centrifuged at 1000 rpm for 5 minutes.
- Supernatent was discarded, and a staining mix of Propidium iodide was added.
- The cell suspension was incubated, and preserved.
Flow cytometry is an effective method for assessing cell populations on the basis of fluorescence as well as light scattering characteristics. In this study, the flow cytometry test will be employed to determine apoptosis within cells with leukemia treatment via the combinational treatment. Following the procedure, the cells will be labeled with Annexin V as well as Propidium Iodide (PI) in order to distinguish between early, late, and necrotic cells. A flow cytometer will be used to examine the fluorescence generated by the labeled cells. This test will offer information on the combination’s effect on leukemia cells’ dying pathways.
2.1.3 Western blotting
- Required amount of cell culture was taken and then suspended into a suspension buffer. Mostly, buffer containing NaCl solution.
- The sample was centrifuged at 5000 rpm for 10 minutes, and the cell pellet was resuspended into the buffer solution (Rickard et al. 2023).
- Then, different concentrations of the sample were taken and loaded into the stacks of microplate of PIERCE fast western blotting kit.
- Next, 10 ml of western antibody diluent was taken and added in the required amount into each stack containing cell suspension.
- After that, 1 ml of Rabbit optimizes HRP reagent was taken and added in the required amount into the stacks.
- At last, the absorbance was taken.
Western blotting provides a method for detecting and quantifying particular proteins in a specimen. It will be used in this study to analyze changes in the expression of proteins in leukemia cells following treatment with MDIVI-1, a drug called or both. Cell lysates are going to be produced and electrophoresis upon a gel made of polyacrylamide will be performed. The proteins are going to be placed on a membrane as well as probed with antibodies specific to the target proteins of concern. HRP-conjugated secondary antibodies as well as a chemiluminescent reagent will be used for detection. Western blotting will reveal important information on adjustments to apoptotic as well as the survival of cell pathways caused by the combinational treatment.
Drug
Doxorubicin is one of the most common and effective anti-cancer representatives. This drug is mostly associated with negative cardiac effects, incorporating progressive heart failure and cardiomyopathy. The study highlighted numerous advantageous effects of the “mitochondrial division inhibitor (mdivi-1)” in a mixture of pathological infections including heart failure and reperfusion and injury, ischemia. The study analyzed the consequences of mdivi-1 on doxorubicin-induced cardiac dysfunction in various stressed conditions within the cell (Dadson et al. 2020). To assess the consequences of drug therapies on the hyper contracture of cardiac myocytes and mitochondrial depolarization. MDIVI-1, a specific regulator of mitochondrial division, showed anti-cancer potential. Mitochondria are essential for cellular survival as well as metabolism, and disruption of mitochondrial dynamics within cells with cancer can result in apoptosis or programmed cell death.
Mechanism of action
In general, the drug doxorubicin works by mixing into the replication mechanism of the cell. The drug intercalated into the DNA, which helps to disrupt the replication cycle of the cellular genome. The drug binds with DNA through its anthraquinone ring. This stabilizes the complex that is formed after the interaction by hydrogen bonds with the bases of the DNA. As a result of the formation of the complex structure, there was a creation of torsional stress within the DNA structure. This resulted in a replacement of individual bases and eviction of nucleosome.
Also, the DNA-doxorubicin complex would block the activity of the enzyme “topoisomerase II”. It does by the prevention of regulation of the breakage of DNA mediated by topoisomerase II. As a result, processes of replication and transcription of the cell hampered, which breaks the cell cycle.
Structure of the drug
Doxorubicin is an anthracyclin antibiotic agent, which is a member of “tetraquinone” family of drugs. The structure of the drug is a primary “alpha-hydroxy ketone” and “tertiary alpha-hydroxy ketone”. The moleculer formula is C27H29NO11.
Symptoms of Leukemia
The symptoms of leukemia were given below.
- Persistent fever for a long period of time.
- Patients were experienced with weakness and fatigue.
- Patients tend to lose weight rapidly.
- Enlarged liver and spleen, swollen lymph nodes.
- Tenderness and pain in the bones, throughout the disease period.
- Appearance of tiny red spots in the skin.
- Bruising or early bleeding in the affected patients.
Current treatment
The treatment that is being selected for this experiment is the combinational therapy of MDIVI-1 and doxorubicin for the treatment of leukemia. The HL60 cell lines were usesd for the evaluation of the effect of pharmacological inhibition of mitochondrial division on the cytotoxicity of doxorubicin in a cell line effected by cancer.
Previous treatment
Previously, there were other methods like chemotherapy, radiotherapy were used. Radiotherapy played important role in the destruction of cancer effected cells.
Reference List
Journals
Anderson, R.G., 2021. Targeting Mitochondria in Resistant Acute Myeloid Leukemia (Doctoral dissertation, Wake Forest University).
Dadson, K., Calvillo-Argüelles, O., Thavendiranathan, P. and Billia, F., 2020. Anthracycline-induced cardiomyopathy: cellular and molecular mechanisms. Clinical science, 134(13), pp.1859-1885.
dos Santos Arruda, F., Tomé, F.D., Miguel, M.P., de Menezes, L.B., Nagib, P.R., Campos, E.C., Soave, D.F. and Celes, M.R.N., 2019. Doxorubicin-induced cardiotoxicity and cardioprotective agents: classic and new players in the game. Current Pharmaceutical Design, 25(2), pp.109-118.
Esmaeili, S.A., Sahranavard, S., Salehi, A. and Bagheri, V., 2021. Selectively targeting cancer stem cells: Current and novel therapeutic strategies and approaches in the effective eradication of cancer. IUBMB life, 73(8), pp.1045-1059.
García-Heredia, J.M. and Carnero, A., 2020. Role of mitochondria in cancer stem cell resistance. Cells, 9(7), p.1693.
Genovese, I., Carinci, M., Modesti, L., Aguiari, G., Pinton, P. and Giorgi, C., 2021. Mitochondria: insights into crucial features to overcome cancer chemoresistance. International journal of molecular sciences, 22(9), p.4770.
Liang, X., Wang, S., Wang, L., Ceylan, A.F., Ren, J. and Zhang, Y., 2020. Mitophagy inhibitor liensinine suppresses doxorubicin-induced cardiotoxicity through inhibition of Drp1-mediated maladaptive mitochondrial fission. Pharmacological Research, 157, p.104846.
Rickard, B.P., Overchuk, M., Obaid, G., Ruhi, M.K., Demirci, U., Fenton, S.E., Santos, J.H., Kessel, D. and Rizvi, I., 2023. Photochemical Targeting of Mitochondria to Overcome Chemoresistance in Ovarian Cancer. Photochemistry and Photobiology, 99(2), pp.448-468.
Song, C., Pan, S., Zhang, J., Li, N. and Geng, Q., 2022. Mitophagy: A novel perspective for insighting into cancer and cancer treatment. Cell Proliferation, 55(12), p.e13327.
TarvijEslami, S., Nasirian, H., Nasirian, A., Abouali, B., Ivrigh, S.R., Arabi, M. and Bereimipour, A., 2023. Selection an Effective Drug Regimen on the Tumor Microenvironment of Non-invasive to Invasive Human Retinoblastoma.
Yun, C.W., Jeon, J., Go, G., Lee, J.H. and Lee, S.H., 2020. The dual role of autophagy in cancer development and a therapeutic strategy for cancer by targeting autophagy. International Journal of Molecular Sciences, 22(1), p.179.
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