A system for evaluating the anticancer effect of a magnesium complex {[Mg(1,10-Phenanthroline)(H2O)4](1,3-Benzthiazol-2-thiolate)2,1,10-Phenanthroline} against squamous cell carcinomas of the oral cavity

Abstract

A system for evaluating the anticancer activity of a magnesium complex {[Mg(1,10-Phenanthroline)(H2O)4](1,3-Benzthiazol-2-thiolate) 2. 1,10-Phenanthroline} against oral squamous cell carcinoma, including: a cell culture apparatus configured to maintain human oral squamous cell carcinoma cells in a controlled environment with specific temperature and CO2 conditions; a compound delivery module configured to deliver predetermined concentrations of the magnesium complex to the cultured cells; a cell analysis unit configured to measure and analyze cellular responses to the magnesium complex; a flow cytometry device configured to analyze cellular parameters such as apoptosis and cell cycle progression; and a data processing unit configured to calculate and analyze cytotoxicity data and to generate inhibitory percentages.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a system for evaluating the anticancer activity of a magnesium complex {[Mg(1,10-phenanthroline)(H2O)4](1,3-benzthiazole-2-thiolate)2,1,10-phenanthroline} against oral squamous cell carcinoma. BACKGROUND OF THE INVENTION

[0002] Metal-based compounds, especially platinum-based drugs such as cisplatin, are crucial in cancer therapy due to their ability to bind DNA and induce cell death. However, drug resistance and severe side effects such as nephrotoxicity and neurotoxicity have limited their effectiveness. This has driven research into the development of new metal-based therapeutic compounds.

[0003] Oral squamous cell carcinoma (OSCC) accounts for 90% of all oral cancers and is the sixth most common cancer worldwide. In Pakistan, it is the second most common cancer. Over 16,000 new cases and 10,000 deaths were reported in 2020, highlighting the urgent need for new treatment options.

[0004] While transition metals have been extensively studied for cancer treatment, alkaline earth metals such as magnesium are still understudied. Magnesium is the fourth most abundant cation in biological systems and essential for hundreds of enzymatic processes. This invention focuses on a novel magnesium complex {[Mg(1,10-phenanthroline)(H,O)4](1,3-benzothiazole-2-thiolate)} 2. 1,10-Phenanthroline}, which combines the following properties: • 1,10-Phenanthroline: A bidentate ligand known for its DNA / RNA cleavage activity • 1,3-Benzothiazol-2-thiol: A natural compound with multiple donor atoms • Magnesium: An essential biological metal

[0005] This complex has previously been known for its anti-urease activity, and this invention explores its potential as an anti-cancer agent specifically targeting OSCC while ensuring safety for normal cells.

[0006] To evaluate the anticancer activity of a magnesium complex, the present invention provides a system for evaluating the anticancer activity of a magnesium complex against oral squamous cell carcinoma. SUMMARY OF THE INVENTION

[0007] The present disclosure relates to a system for evaluating the anticancer activity of a magnesium complex {[Mg(1,10-phenanthroline)(H2O)4] (1,3-benzthiazole-2-thiolate) 2.1,10-Phenanthroline} against oral squamous cell carcinoma. This invention presents a novel magnesium-based complex {[Mg(1,10-Phenanthroline)(H2O)4] (1,3-Benzthiazole-2-thiolate) 2.1,10 Phenanthroline as a therapeutically effective anticancer agent specifically targeting oral squamous cell carcinoma (OSCC). The system combines cell analysis, flow cytometry, and molecular biology techniques to evaluate the compound's anticancer properties while ensuring its safety for normal cells.

[0008] The present disclosure is intended to provide a system for evaluating the anticancer activity of a magnesium complex {[Mg(1,10-phenanthroline)(H2O)4](1,3-benzthiazole-2-thiolate) 2.1,10-Phenanthroline} against oral squamous cell carcinoma. The system comprises: a cell culture device configured to maintain human oral squamous cell carcinoma cells in a controlled environment with specific temperature and CO2 conditions; a compound delivery module configured to deliver predetermined concentrations of the magnesium complex to the cultured cells; a cell analysis unit configured to measure and analyze cellular responses to the magnesium complex; a flow cytometry device configured to analyze cellular parameters including apoptosis and cell cycle progression; and a data processing unit configured to calculate and analyze cytotoxicity data and generate inhibition percentages.

[0009] An object of the present disclosure is to provide a system for evaluating the anticancer activity of a magnesium complex {[Mg(1,10-phenanthroline)(H2 O)4](1,3-benzthiazole-2-thiolate) 2. 1,10-Phenanthroline} against oral squamous cell carcinoma

[0010] Another objective of the present disclosure is to develop and evaluate a novel magnesium complex that effectively targets OSCC cells while exhibiting minimal toxicity to normal cells.

[0011] Another objective of the present disclosure is a comprehensive system for analyzing anticancer properties, including cytotoxicity, apoptosis induction, and antimigration effects.

[0012] To further clarify the advantages and features of the present disclosure, a more detailed description of the invention will be given with reference to specific embodiments thereof, which are illustrated in the accompanying drawings. It should be noted that these drawings represent only typical embodiments of the invention and are therefore not to be considered limiting its scope. The invention will be described and explained in additional detail and in greater detail with reference to the accompanying drawings. SHORT DESCRIPTION OF THE FIGURE

[0013] These and other features, aspects, and advantages of the present disclosure will be better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. Where: Fig. Figure 1 shows a block diagram of a system for evaluating the anticancer activity of a magnesium complex {[Mg(1,10-phenanthroline)(H2 O)4](1,3-benzthiazole-2-thiolate) 2. 1,10-phenanthroline} against oral squamous cell carcinoma according to an embodiment of the present disclosure.

[0014] Furthermore, those skilled in the art will appreciate that elements in the drawings are shown for convenience and may not necessarily be drawn to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Furthermore, with respect to device construction, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawing may show only the specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawings with details that would be readily apparent to those skilled in the art who would benefit from the description herein. DETAILED DESCRIPTION:

[0015] To facilitate an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and described in specific language. It is to be understood, however, that no limitation on the scope of the invention is intended thereby, since such changes and further modifications of the illustrated system, and such further applications of the principles of the invention as illustrated therein, are contemplated as would normally occur to one skilled in the art to which the invention pertains.

[0016] It will be understood by one skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.

[0017] References in this specification to "one aspect," "another aspect," or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, occurrences of the phrase "in one embodiment," "in another embodiment," and similar language throughout this specification may or may not all refer to the same embodiment.

[0018] The terms "comprises," "comprising," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps not only includes those steps, but may also include other steps not expressly listed or inherent in such process or method. Likewise, one or more devices or subsystems or elements or structures or components preceded by "comprises...a" does not preclude, without further limitation, the existence of other devices or other subsystems or other elements or other structures or other components or additional devices or additional subsystems or additional elements or additional structures or additional components.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are for illustrative purposes only and should not be considered limiting.

[0020] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0021] Fig. Figure 1 shows a block diagram of a system (100) for evaluating the anticancer activity of a magnesium complex {[Mg(1,10-phenanthroline)(H2O)4](1,3-benzthiazole-2-thiolate) 2. 1,10-phenanthroline} against oral squamous cell carcinoma according to an embodiment of the present disclosure.

[0022] With reference to Fig.1, the system (100) comprises: a cell culture device (102) configured to maintain human oral squamous cell carcinoma cells in a controlled environment with specific temperature and CO2 conditions; a compound delivery module (104) configured to deliver predetermined concentrations of the magnesium complex to the cultured cells; a cell analysis unit (106) configured to measure and analyze cellular responses to the magnesium complex; a flow cytometry device (108) configured to analyze cellular parameters including apoptosis and cell cycle progression; and a data processing unit (110) configured to calculate and analyze cytotoxicity data and generate inhibition percentages.

[0023] In one embodiment, the cell culture device (102) comprises: an incubator (102a) maintained at 37°C with 5% CO2; and culture vessels (102b) selected from 96-well plates, 24-well plates, and 6-well plates, wherein the device uses a growth medium comprising Dulbecco's modified Eagle's medium supplemented with fetal bovine serum and antibiotics.

[0024] In one embodiment, the cell analysis unit (106) comprises: a spectrophotometer configured to measure absorbance at 570 nm; an inverted phase contrast microscope configured to detect cellular morphological changes; and a fluorescence microscope configured to analyze nuclear morphology using DAPI staining.

[0025] In one embodiment, the compound administration module (104) is configured to: prepare serial dilutions of the magnesium complex, maintain specified treatment durations, and administer concurrent control treatments, including cisplatin.

[0026] In one embodiment, the flow cytometry device (108) comprises: multiple laser excitation sources; fluorescence detection channels for FITC and PI; and an analysis module configured to process cellular parameters including apoptosis and cell cycle distribution.

[0027] In one embodiment, the system (100) further comprises: a migration analysis module (112) configured to generate and measure standardized scratches in cell monolayers, monitor cell migration rates, and calculate the percentage closure of scratches over time.

[0028] In one embodiment, the system (100) further comprises: an invasion analysis module (114) comprising: cell culture inserts with defined pore sizes; a matrix coating device; and imaging systems for quantifying cell invasion.

[0029] In one embodiment, the system (100) further comprises: a gene expression analysis module (116) configured to extract RNA from treated cells, synthesize cDNA, and perform quantitative PCR analysis.

[0030] In one embodiment, the system (100) further comprises: an immunocytochemistry module (118) configured to fix and permeabilize treated cells, apply primary and secondary antibodies, and detect and analyze protein expression patterns.

[0031] In one embodiment, the data processing unit (110) comprises a dedicated processor (110a) configured to calculate IC50 values; perform statistical analyses; generate dose-response curves; and create graphical representations of the results.

[0032] The present invention relates to a sophisticated system for evaluating a novel magnesium complex as an anticancer agent. At its core, the system utilizes specialized cell culture conditions in which human oral squamous cell carcinoma cells (CAL 27) and normal human fibroblast cells (BJ) are maintained in controlled environments. The cells are exposed to various concentrations of the magnesium complex under precise conditions, with cellular responses monitored by multiple analytical methods. The system utilizes advanced spectrophotometric analysis to measure cytotoxicity through MTT assays and provides quantitative data on cell viability and IC50 values. Morphological changes are monitored using phase-contrast and fluorescence microscopy, allowing detailed observation of treatment-induced cellular and nuclear changes.The system includes flow cytometry analysis for the precise measurement of apoptosis induction, changes in mitochondrial membrane potential, and cell cycle distribution. A key feature of the system is its ability to assess the compound's effect on cancer cell migration and invasion. This is achieved through specialized assays, including scratch assays, migration chambers, and matrix invasion studies. The system also includes molecular analysis components for investigating changes in gene expression and protein modulation using RT-PCR and immunocytochemistry. The safety assessment component of the system includes in vivo toxicity analysis in animal models with comprehensive histopathological examination of vital organs.All generated data are processed by dedicated software for statistical analysis and result interpretation, ensuring a reliable and reproducible assessment of the compound's anticancer potential.

[0033] The human oral squamous cell carcinoma cell line (CAL 27; CRL 2095) and the human normal fibroblast cell line (BJ; CRL-2522) were cultured and maintained in a controlled environment. The system consisted of Dulbecco's modified Eagle's medium (DMEM) with high glucose supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. Incubation conditions were set at 37°C with 5% CO2 to ensure optimal cell growth and maintenance.

[0034] To determine the cytotoxicity of the magnesium complex, an MTT assay was performed. CAL 27 and BJ cells were cultured in a 96-well plate at a density of 1.2 × 10 4or 1 × 10 4 Cells were seeded per well and grown until 80% confluency was reached. Cell densities were adjusted based on the cell doubling time to maintain a uniform monolayer. After cell growth, treatments with different concentrations of the magnesium complex and cisplatin were administered for 48 hours, while control cells remained untreated. After treatment, the medium was removed, and the cells were incubated with a 0.5 mg / ml MTT solution for 4 hours. Formazan crystals formed in viable cells were solubilized with dimethyl sulfoxide (DMSO), and absorbance at 570 nm was recorded using a BioTek Synergy HTX Reader. Percent inhibition was calculated using a standard formula, and IC50 values ​​were determined using EZ-Fit Enzyme Kinetics software.

[0035] For analysis of morphological changes, CAL 27 cells were cultured in a 6-well plate at a density of 3.5 × 104 pro Wells were seeded and incubated overnight. The monolayer was treated with 1.7, 2, and 2.3 µM of the magnesium complex and 12 and 2 µM of cisplatin. Untreated cells served as controls. Morphological changes were observed and recorded after 0, 24, and 48 hours using an inverted phase-contrast microscope at 20× magnification.

[0036] To assess nuclear morphology, CAL 27 cells were cultured at a density of 9 × 104 Zellenper well on coverslips in a 24-well plate and incubated overnight. After treatment with 1.7, 2, and 2.3 µM of the magnesium complex for 48 hours, control cells remained untreated. The cells were fixed with 3.7% formaldehyde for 10 minutes, stained with DAPI for 10 minutes, and mounted with Mowiol Mounting Medium. Imaging was performed after 24 and 48 hours using an Eclipse NiE microscope at 20x magnification.

[0037] To evaluate the antimigratory effect, a scratch test was performed. CAL 27 cells were cultured in a 6-well plate at a density of 2.5 × 10 3Cells were seeded per well and incubated overnight. A scratch was introduced into the monolayer, and the wells were washed with phosphate-buffered saline. Treatment was administered with 1.7, 2, and 2.3 µM of the magnesium complex and 12 and 2 µM of cisplatin, while control cells remained untreated. Images of the scratch area were captured using an inverted phase-contrast microscope at 10x magnification. The percentage closure of the scratch was calculated using the system's data processing unit.

[0038] For the migration test, cell culture inserts with a pore size of 0.8 µm were placed in a 24-well plate. CAL 27 cells (6 × 10 4) were placed in inserts in DMEM without FBS and treated with 1.7, 2, and 2.3 µM of the magnesium complex or 12 and 2 µM cisplatin. Control cells remained untreated. The lower chamber contained DMEM with 10% FBS, and the plates were incubated for 48 hours. After incubation, the medium was removed, and migrated cells were fixed with 3.7% formaldehyde and stained with 0.1% crystal violet. Imaging was performed using an Eclipse Ti2 inverted microscope at 4× magnification.

[0039] To assess invasion potential, inserts coated with Geltrex matrix were used. The inserts were coated with 80 µl of Geltrex and incubated for 1 hour at 37°C. CAL 27 cells (2 × 10^5) were suspended in DMEM without FBS containing 1.7, 2, and 2.3 µM of the magnesium complex or 12 and 2 µM of cisplatin and seeded into the upper chamber of the insert. Control cells remained untreated. The lower chamber contained DMEM with 10% FBS, and the plates were incubated for 48 hours. After incubation, migrated cells were fixed with 3.7% formaldehyde and stained with 0.1% crystal violet. Imaging was performed using an Eclipse Ti2 inverted microscope at 4× magnification.

[0040] The clonogenicity assay was performed to evaluate the long-term effect of the magnesium complex on cell attachment and growth. CAL 27 cells (1 × 106) were seeded into a 6-well plate and treated with 1.7, 2, and 2.3 µM of the magnesium complex for 48 hours. Control cells remained untreated. After treatment, the cells were trypsinized, counted with trypan blue dye, and reseeded at the same density in a 6-well plate for 15 days of incubation. Colonies were then fixed with 3.7% formaldehyde and stained with 0.1% crystal violet.

[0041] To determine apoptosis, a membrane permeability / apoptosis assay of dead cells was performed using YO-PRO-1 and PI dyes. CAL 27 cells (1 × 10^6) were seeded into a 6-well plate and incubated overnight. The monolayer was treated with 1.7, 2, and 2.3 µM of the magnesium complex for 48 hours, while control cells remained untreated. After treatment, the cells were harvested, washed three times with PBS, and stained for 30 minutes with 1 µL each of YO-PRO-1 and PI dyes. The analysis was performed using a BD FACSCelesta flow cytometer on FITC and PI channels. The data were analyzed using the system's data processing unit.

[0042] The system is configured to determine the effect of a magnesium complex on mitochondrial membrane potential using flow cytometry. To determine the effects, 1 × 10 6CAL 27 cells are seeded into a 6-well plate and incubated overnight. The monolayer is treated with 1.7, 2, and 2.3 µM of the magnesium complex and incubated for 48 hours. Untreated control cells remain untreated under the same conditions. After the incubation period, the cells are harvested and collected in centrifuge tubes. A positive control is created by treating the cells with 2 µl of CCCP and incubating them for 10 minutes at 37°C. All samples are stained with 2 µM JC-1 dye for 30 minutes. The cells are then pelleted by centrifugation and resuspended in 500 µl of PBS. The system includes a flow cytometer configured for sample analysis using FITC and PI channels. Data processing is performed using the system's data processing unit.

[0043] The system also evaluates the cell cycle modulating effects of the magnesium complex. For this purpose, 2 × 10 6CAL 27 cells were seeded into a 6-well plate and incubated overnight. The monolayer was treated with 1.7, 2, and 2.3 µM of the magnesium complex and incubated for 48 hours. Control cells remained untreated. After treatment, the cells were harvested and collected in centrifuge tubes. The samples were washed with PBS and fixed overnight at 4°C with chilled 70% ethanol. The ethanol solution was added dropwise while vortexing to prevent clumping. After fixation, the cells were pelleted at 300 x g for 30 minutes and washed three times with PBS. The system processed the samples by treating them with 0.5 µg / ml DNase-free RNase solution and then incubating them for 2 hours at 37°C. Subsequently, 50 µg / ml PI was added, and the samples were incubated for 1 hour at room temperature. The flow cytometer is used for cell cycle analysis on the PI channel and the data processing unit processes the data.

[0044] The system also includes a gene expression analysis module by 1 × 10 6 CAL 27 cells per well in a 6-well plate were treated with 1.7 and 2 µM of the magnesium complex for 48 hours. Control cells remained untreated. Total RNA was extracted using a reagent according to the manufacturer's protocol. This procedure involved treating the cells with the reagent and chloroform to separate the aqueous phase, followed by RNA purification with isopropyl alcohol and 75% ethanol. The RNA samples were then transcribed into cDNA using a First Strand cDNA Synthesis Kit. The synthesized cDNA served as a template for gene expression analysis using Maxima SYBR Green / ROX qPCR Master Mix along with forward and reverse primers of the target gene.

[0045] The system is configured for immunocytochemistry. CAL 27 cells are seeded in a 48-well plate at a density of 40,000 cells per well and incubated overnight. The cells are then treated with 1.7 and 2 µM of the magnesium complex for 48 hours, while the control cells remain untreated. Following treatment, the cells are washed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes. After washing with PBS, the cells are incubated for 1 hour at 37°C with a blocking solution consisting of 2% bovine serum albumin and 0.1% Tween 20 in PBS. The system allows the use of primary antibodies, which are incubated overnight at 4°C, then washed for 1 hour at room temperature and treated with secondary antibodies.DAPI staining is performed and images are taken under an Eclipse Ti2 microscope at 20x magnification.

[0046] The system includes a module for assessing acute toxicity using Balb / c mice of both sexes, randomly divided into four groups. The magnesium complex is dissolved in drinking water containing 4% DMSO. The control group receives only the vehicle, while the treatment groups receive a single oral dose of 10, 50, and 250 mg / kg body weight. The system continuously monitors behavioral changes for three hours, followed by half-hourly observations for the next four hours. Mortality is monitored until the fourth day after administration. Following the study, organs such as the kidney and liver are surgically removed, fixed in 4% formaldehyde, and gradually dehydrated with 70%, 90%, and 100% ethanol. The system processes tissue samples by incubating them in a 1:1 ratio of xylene and paraffin for one hour, followed by incubation in paraffin alone overnight at 65°C.Tissue sections of 5-6 micrometers are prepared on slides, rehydrated stepwise with decreasing ethanol concentrations (100%, 90%, and 70%), and stained with hematoxylin and eosin Y dyes. The slides are mounted with DPX and visualized under a bright-field microscope at 10x magnification.

[0047] The system's data processing unit is also configured to perform statistical analyses of the data obtained and then present the results in a graphical representation.

[0048] The results showed that the synthesized magnesium complex exhibited potent anticancer activity against OSCC cells (CAL 27) while exhibiting significantly lower toxicity to normal human cells (BJ cells). It effectively induced apoptosis by disrupting the mitochondrial membrane potential, causing DNA condensation, and driving the cells into the late apoptotic stage. The complex arrested the cell cycle in the G1 phase, reduced clonogenic potential, and significantly inhibited migration and invasion. At the molecular level, the treatment downregulated key oncogenic genes and proteins, including WNT, β-catenin, c-MYC, COX-2, RAS, MMP-2, EGFR, ERK, and VEGF-A, while upregulating pro-apoptotic markers such as BAX, caspase-3, and E-cadherin. This modulation of gene and protein expression suppressed the proliferation, migration and angiogenesis of cancer cells.In vivo toxicity studies showed no significant adverse effects on liver and kidney tissue, confirming its safety at higher doses. These results suggest that the magnesium complex is a promising anticancer agent, with selective cytotoxicity against OSCC cells, minimal toxicity to normal cells, and potent pro-apoptotic and antimetastatic properties.

[0049] The results suggest that the magnesium complex is a novel metal-based anticancer compound that demonstrated potent cytotoxic and antimigratory effects against oral squamous cell carcinoma (OSCC) in vitro, while remaining non-toxic to normal human fibroblast cells. The complex significantly induced apoptosis and reduced the clonogenicity, migratory, and invasive potential of cancer cells. The treatment also modulated the expression of apoptotic and metastatic genes and proteins. The complex showed no major signs of toxicity in the acute in vivo toxicity analysis in mice. Taken together, the data suggest that the magnesium complex may be a potential drug candidate for OSCC therapy.

[0050] The drawings and the foregoing description provide examples of embodiments. Those skilled in the art will recognize that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of the processes described herein may be changed and are not limited to the manner described herein. Furthermore, the actions of any flowchart need not be implemented in the order shown; nor do all actions necessarily need to be performed. Also, those actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is in no way limited by these specific examples.Numerous variations, whether explicitly stated in the specification or not, such as differences in structure, dimensions, and material use, are possible. The scope of the embodiments is at least as broad as indicated in the following claims.

[0051] Advantages, other benefits, and solutions to problems have been described above with reference to specific embodiments. However, the advantages, benefits, solutions to problems, and any components that may cause an advantage or solution to occur or become more apparent are not to be construed as a critical, required, or essential feature or component of any or all of the claims. REFERENCES 100 A system for evaluating the anticancer activity of a magnesium complex {[Mg(1,10-phenanthroline)(H2O)4]1(1,3-benzthiazole-2-thiolate)2,1,10-phenanthroline} against oral squamous cell carcinoma. 102 cell culture devices 102a Incubator 102b Culture vessels 104 Connection Management Module 106 Cellular Analysis Unit 108 Flow cytometry device 110 Data processing unit 110a Dedicated Processor 112 Migration Analysis Module 114 Invasion Analysis Module 116 Gene Expression Analysis Module 118 Immunocytochemistry Module

Claims

[1] A system for evaluating the anticancer activity of a magnesium complex {[Mg(1,10-phenanthroline)(H2O)4](1,3-benzthiazole-2-thiolate) 2. 1,10-Phenanthroline} against oral squamous cell carcinoma, comprising: a cell culture apparatus configured to maintain human oral squamous cell carcinoma cells in a controlled environment with specific temperature and CO2 conditions; a compound delivery module configured to deliver predetermined concentrations of the magnesium complex to the cultured cells; a cell analysis unit configured to measure and analyze cellular responses to the magnesium complex; a flow cytometry device configured to analyze cellular parameters such as apoptosis and cell cycle progression; and a data processing unit configured to calculate and analyze cytotoxicity data and generate inhibition percentages. [2] The system of claim 1, wherein the cell culture device comprises: an incubator maintained at 37°C with 5% CO2; and culture vessels selected from 96-well plates, 24-well plates, and 6-well plates, the device using a growth medium containing Dulbecco's modified Eagle's medium supplemented with fetal bovine serum and antibiotics. [3] The system of claim 1, wherein the cell analysis unit comprises: a spectrophotometer configured to measure absorbance at 570 nm; an inverted phase contrast microscope configured to detect cellular morphological changes; and a fluorescence microscope configured to analyze nuclear morphology using DAPI staining. [4] The system of claim 1, wherein the compound delivery module is configured to: prepare serial dilutions of the magnesium complex, maintain specified treatment durations, and concurrently administer control treatments, including cisplatin. [5] The system of claim 1, wherein the flow cytometry device comprises: multiple laser excitation sources; fluorescence detection channels for FITC and PI; and analytical software configured to process cellular parameters including apoptosis and cell cycle distribution. [6] The system of claim 1, further comprising: a migration analysis module configured to generate and measure standardized scratches in cell monolayers, monitor cell migration rates, and calculate the percentage closure of scratches over time. [7] The system of claim 1, further comprising: an invasion analysis module comprising: cell culture inserts with defined pore sizes; a matrix coating device; and imaging systems for quantifying cell invasion. [8] The system of claim 1, further comprising: a gene expression analysis module configured to extract RNA from treated cells, synthesize cDNA, and perform quantitative PCR analysis. [9] The system of claim 1, further comprising: an immunocytochemistry module configured to fix and permeabilize treated cells, apply primary and secondary antibodies, and detect and analyze protein expression patterns. [10] The system of claim 1, wherein the data processing unit comprises a dedicated processor configured to: calculate IC50 values; perform statistical analyses; generate dose-response curves; and create graphical representations of the results.

Images